Synthesis and evaluation of CS-2100, a potent, orally active and S1P 3- sparing S1P1 agonist

Article abstract of DOI:10.1016/j.ejmech.2012.02.022

Modulators of sphingosine phosphate receptor-1 (S1P₁) have recently been focused as a suppressant of autoimmunity. We have discovered a 4-ethylthiophene-based S1P₁ agonist 1-({4-Ethyl-5-[5-(4- phenoxyphenyl)-1,2,4-oxadiazol-3-yl]-2-t

Full text of DOI:10.1016/j.ejmech.2012.02.022

European Journal of Medicinal Chemistry 51 (2012) 92e98
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of CS-2100, a potent, orally active and S1P3- sparing S1P1
agonist
Tsuyoshi Nakamura a, Masayoshi Asano a, Yukiko Sekiguchi a, Yumiko Mizuno a, Kazuhiko Tamaki a,
Futoshi Nara b, Yumi Kawase b, Yoshiyuki Yabe c, Daisuke Nakai c, Emi Kamiyama c, Yoko Urasaki-Kaneno c,
Takaichi Shimozato d, Hiromi Doi-Komuro d, Takashi Kagari d, Wataru Tomisato d, Ryotaku Inoue d,
,
Miyuki Nagasaki d, Hiroshi Yuita e, Keiko Oguchi-Oshima e, Reina Kaneko f, 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 Cardiovascular-Metabolics Research Laboratories, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
c Drug Metabolism & Pharmacokinetics Research Laboratories, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
d Frontier Research Laboratories, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
e Oncology Research Laboratories, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
f Global Project Management Department, 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:
Modulators of sphingosine phosphate receptor-1 (S1P1) have recently been focused as a suppressant of
Received 15 December 2011
Received in revised form
4 February 2012
Accepted 13 February 2012
Available online 25 February 2012
autoimmunity. We have discovered
a 4-ethylthiophene-based S1P1 agonist 1-({4-Ethyl-5-[5-(4-
phenoxyphenyl)-1,2,4-oxadiazol-3-yl]-2-thienyl}methyl)azetidine-3-carboxylic acid (CS-2100, 8)
showing potent S1P1 agonist activity against S1P3 and an excellent in vivo potency. We report herein the
synthesis of CS-2100 (8) and pharmacological effects such as S1P1 and S1P3 agonist activity in vitro,
peripheral blood lymphocyte lowering effects and the suppressive effects on adjuvant-induced arthritis
and experimental autoimmune encephalomyelitis (EAE) in animal models. The pharmacokinetic data
were also reported. CS-2100 (8) had >5000-fold greater agonist activity for human S1P1 (EC50; 4.0 nM)
relative to S1P3 (EC50; >20000 nM). Following administration of single oral doses of 0.1 and 1 mg/kg of
CS-2100 (8) in rats, lymphocyte counts decreased significantly, with a nadir at 8 and/or 12 h post-dose
and recovery to vehicle control levels by 24e48 h post-dose. CS-2100 (8) is efficacious in the adjuvant-
induced arthritis model in rats (ID50; 0.44 mg/kg). In the EAE model compared to the vehicle-treated
group, significant decreases in the cumulative EAE scores were observed for 0.3 and 1 mg/kg CS-2100
(8) groups in mice. While CS-2100 (8) showed potent efficacy in various animal disease models, it was
also revealed that the central 1,2,4-oxadiazole ring of CS-2100 (8) was decomposed by enterobacteria in
intestine of rats and monkeys, implicating the latent concern about an external susceptibility in its
metabolic process in the upcoming clinical studies.
Keywords:
S1P1
Agonist
CS-2100
Lymphocyte
Adjuvant-induced arthritis
EAE
Multiple sclerosis
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
lymphocyte trafficking [1e4]. Knowledge of the physiology and
pharmacology of S1P1 has evolved rapidly through studies
Sphingosine 1-phosphate (S1P) receptor-1 (S1P1) is one of five
G-proteinecoupled receptors which bind S1P, a bioactive lipid
mediator involved in cell differentiation, morphogenesis, angio-
genesis, motility, and multiple other processes [1,2]. Modulation of
S1P receptors may have important clinical applications. In partic-
ular, targeting S1P1 with synthetic modulators appears to have
clinical utility in the suppression of autoimmunity by affecting
involving Gilenya (fingolimod, FTY720) (1), which was approved for
the treatment of patients with relapsing remitting multiple scle-
rosis (RRMS) [5,6]. According to previous reports, the active phos-
phorylated form of fingolimod, FTY720-P (2) [7e9], which is
generated in vivo via a sphingosine kinase, acts as an S1P1 agonist or
functional antagonist. However, FTY720-P (2) lacks specificity due
to its affinity for S1P3, S1P4, and S1P5 [10,11]. The beneficial effects
of fingolimod are thought to be mediated via S1P1. On the other
hand, S1P3 is reported to be concerned with bradycardia in rodents
[12e14]. Recent studies suggested that only removal of the S1P3
agonism is not sufficient to exclude the cardiovascular side effect
* Corresponding author. Tel.: þ81 3 3492 3131; fax: þ81 3 5436 8563.
E-mail address: nishi.takahide.xw@daiichisankyo.co.jp (T. Nishi).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.02.022

T. Nakamura et al. / European Journal of Medicinal Chemistry 51 (2012) 92e98
93
[15]. However, sparing the S1P3 agonism has been proposed as
a medicinal chemical approach to reduce potential side effects. We
therefore focused our attention on synthesizing compounds with
greater S1P1/S1P3 selectivity. Comprehensive evaluation of various
properties of these synthesized compounds led to the identification
of a promising clinical candidate, CS-0777 (3), conclusively [16]. CS-
0777 is currently undergoing human clinical trials, and the initial
clinical results for single dose administration of CS-0777 in healthy
subjects and a 12-week open-label study in patients with multiple
sclerosis have also been reported separately [17].
The success of 1 in RRMS clinical trials has encouraged a new
and startling development of S1P1 agonists [18,19]. Especially,
SEW2871 (5), which is screening hits of non-phosphate type S1P1
selective agonist from Scripps and Norvartis, has attracted attention
as the second stream of S1P1 agonists [20,21]. Over the past decade,
a structurally-broad variety of S1P1 agonists have been disclosed as
exemplified by thiazolo[5,4-b]pyridine 6 (Amgen) [22] or 2-imino-
thiazolidin-4-one 7 (Actelion) [23] in Fig. 1 and other compounds
[14,24e27].
The reduction in peripheral blood lymphocyte counts following
single doses of 1.0 and 2.5 mg CS-0777 (3) was sustained for more
than 2 weeks. This prolonged pharmacologic effect likely reflects
the relatively long pharmacokinetic half-life, although phosphor-
ylation kinetics, selective accumulation of the drug in lymphocytes,
or other factors may play a role. The half-life of CS-0777-P (4) in
blood was w7e8 days [17], similar to that reported for fingolimod
(1). Achieving improved S1P1/S1P3 selectivity and decreased rela-
tive long half-life of CS-0777 (3) has been a consistent theme of our
efforts. In this context, we focused our attention on non-phosphate
type analogues to develop a potent and S1P3-sparing S1P1 agonist
with adequate pharmacokinetic half-life. Among several non-
phosphate type compounds that we explored, 4-ethylthiophene
analogues appeared most promising and they were used for
further scrutinization [28]. We then discovered a novel S1P3-
sparing S1P1 agonist CS-2100 (8), which shows excellent in vivo
potency.
2. Results and discussion
The synthetic route of CS-2100 (8) is shown in Scheme 1. The
cross coupling reaction of silylether 9 [29] with ethylmagnesium
bromide proceeded smoothly by using a catalytic amount of
NiCl2(dppp) to afford ethyl thiophene 10 in good yield. The 5-
position on the thiophene ring was formylated by treatment with
n-BuLi followed by DMF. Aldehyde 11 was treated with hydroxyl-
amine hydrochloride in the presence of triethylamine to give
oxime, which was dehydrated with DCC in toluene to afford nitrile
12. Nitrile 12 was converted to key intermediate amidoxime 13 by
treatment with 40% aqueous hydroxylamine in EtOH. Amidoxime
13 was esterified with 4-phenoxybenzoic acid in the presence of
WSCeHCl and HOBt. Cyclization of the oxadiazole ring and
deprotection of the TBS group proceeded smoothly by treatment
with TBAF in THF to afford alcohol 15 in 89% yield. Alcohol 15 was
treated with CBr4 and PPh3 in dichloromethane, which was then
successively treated with methyl azetidine-3-carboxylate hydro-
chloride in the presence of diisopropylethylamine to afford methyl
ester 16 in good yield. The methyl ester group was saponified with
1 M NaOH in 1,4-dioxane, and then neutralized with AcOH, which
afforded CS-2100 (8) as a white crystalline solid.
Next, agonist-specificity of CS-2100 (8) was determined by
measuring agonist-evoked [35S]GTP
g-S binding activity to rat and
human S1P1 or S1P3 expressed in transfected CHO-K1 cells as
shown in Table 1. The active FTY720-P (2) and CS-0777-P (4) were
included as a comparator. CS-0777-P (4) demonstrated potent
agonist activity for rat and human S1P1 (EC50 ¼ 1.8 and 1.1 nM,
respectively) and at least 100-fold greater activity for S1P3
(EC50 ¼ 200 and 350 nM, respectively). Relative to S1P3 agonist
activity, CS-0777-P (4) appears to have greater S1P1/S1P3 selectivity
HO
Me
RO
N
Me
RO
NH2
O
NH2
Me
1, R = H (Gilenya, fingolimod; FTY720)
2, R = PO3H2 (FTY720-P)
3, R = H (CS-0777)
4, R = PO3H2 (CS-0777-P)
CO2H
F3C
S
O
N
N
N
N
S
N
CF3
F
5 (SEW2871)
6 (AMG 369)
OH
O
N
N
O
CO2H
O
N
OH
S
S
N
N
Cl
O
7 (ponesimod)
8 (CS-2100)
Fig. 1. Structures of FTY720 (1), FTY720-P (2), CS-0777 (3), CS-0777-P (4), SEW2871 (5), AMG 369 (6), ponesimod (7) and CS-2100 (8).

94
T. Nakamura et al. / European Journal of Medicinal Chemistry 51 (2012) 92e98
Br
a
b
c, d
OTBS
OTBS
OTBS
OTBS
OHC
S
S
S
9
10
11
O
NH2
e
f
g
O
N
OTBS
N
HO
S
NC
S
S
O
NH2
OTBS
12
13
14
O
O
N
N
O
O
O
CO2Me
N
N
j
h, i
N
S
S
OH
N
15
16
O
CO2H
N
S
N
8 (CS-2100)
Scheme 1. Reagents and conditions: (a) EtMgBr, cat.NiCl2(dppp), Et2O, 95%; (b) n-BuLi then DMF, THF, 86%; (c) NH2OHeHCl, NEt3, MeOH, CH2Cl2; (d) DCC, toluene, 69% in 2 steps;
(e) aq.NH2OH, EtOH, 60%; (f) 4-Phenoxybenzoic acid, WSCeHCl, HOBt, CH3CN, THF; (g) TBAF, THF, 89% in 2 steps; (h) CBr4, PPh3, CH2Cl2; (i) Methyl azetidine-3-carboxylate-HCl, i-
Pr2NEt, CH2Cl2, 89% in 2 steps; (j) aq.1 M NaOH, 1,4-dioxane, then neutralized with AcOH, 86%.
compared to FTY720-P (2). Compared with CS-0777-P (4), CS-2100
(8) has much higher S1P1/S1P3 selectivity for rat and human S1P1
(EC50 ¼ 1.5 and 4.0 nM, respectively) and at least 5000-fold greater
activity for S1P3 (EC50 ¼ 7400 and >20000 nM, respectively). In
addition, the agonist activity (EC50) of CS-2100 (8) for human S1P4
and S1P5 were 58 and 17 nM, respectively, and it had no ability to
bind human S1P2 (>20000 nM).
In a second series of studies, the effects of CS-2100 (8) on
peripheral blood lymphocyte counts were investigated in rats. In
LEW/CrlCrlj (Lewis) rats administered a single oral dose of 0.1 or
1 mg/kg of CS-2100 (8), the peripheral blood lymphocyte number
decreased to 27% and 11% of vehicle-treated control values for the
0.1 and 1 mg/kg dose levels, respectively, 8 h after CS-2100 (8)
administration (Fig. 2). The decreased peripheral blood lymphocyte
number was recovered to vehicle control levels by 24e48 h post-
dose. These data suggests that CS-2100 (8) exhibits relatively
quick recovery of the peripheral blood lymphocyte number in
Lewis rats compared to CS-0777 (3) (30% and 72% of control for 0.1
or 1 mg/kg of CS-0777 (3) at 48 h oral post-dose) [16].
were administered vehicle (Control), or 0.1, 0.3, 1, or 3 mg/kg CS-
2100 (8) from Day 0 to Day 17, orally once daily. The volumes of
the right hind foot (adjuvant-injected foot) of all rats were
measured on Day 0, 3, 5, 7, 10, 13, 15 and 18 with a plethysmometer
(Fig. 3). A linear regression curve was obtained from the % inhibi-
tion and the logarithmic value of the dosage by least squares
regression analysis, and the ID50 value of the compound was
calculated from the curve. CS-2100 (8) showed a strong suppressive
effect on adjuvant-induced arthritis in rats with 0.44 mg/kg of the
ID50 value on Day 18.
Next, we evaluated the suppressive effect of CS-2100 (8) on
experimental autoimmune encephalomyelitis (EAE) in mice. EAE
has been used as a preclinical animal model for proof of concept
studies for multiple sclerosis therapy. Vehicle-administered mice
began to show clinical signs of EAE from Day 12, and thereafter an
8
7
6
5
4
3
2
1
0
In addition, CS-2100 (8) was evaluated for its suppressive effect
on adjuvant-induced arthritis in Lewis rats. The rats in the normal
group were administered vehicle and the adjuvant-injected rats
Table 1
In vitro agonist-evoked GTPg
-S binding to rat and human S1P1 and S1P3.a
Compound
Rat
EC50 (nM)
Human
Selectivity
(human S1P1 vs S1P3)
0
5
10
15
20
25
30
35
40
45
50
Time after oral administration (h)
S1P1
S1P3
S1P1
S1P3
2 (FTY720-P)
4 (CS-0777-P)
8 (CS-2100)
0.29
1.8
1.5
1.3
200
7400
0.37
1.1
4.0
3.3
350
>20000
9
Fig. 2. Effects of a single oral dose of CS-2100 (8) on lymphocyte counts in rats. Vehicle
(1% MC solution, >), 0.1 mg/kg (C), or 1 mg/kg of CS-2100 (8) (:) was orally
administered to rats. The rats were anesthetized and abdominally dissected 0.5 h, 1 h,
2 h, 4 h, 8 h, 12 h, 24 h, and 48 h after oral administration. Lymphocyte numbers are
shown as mean ꢀ SE.
320
>5000
a
EC50 is defined as the mid-point between the binding ratio of the vehicle and the
maximum response of the test compound.

T. Nakamura et al. / European Journal of Medicinal Chemistry 51 (2012) 92e98
95
2.50
2.00
1.50
1.00
0.50
0.00
1000
Normal
Rat 0.1 mg/kg
Rat 1 mg/kg
Control
0.1 mg/kg CS-2100
0.3 mg/kg CS-2100
1 mg/kg CS-2100
3 mg/kg CS-2100
100
10
Mouse 1 mg/kg
1
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18
Time after adjuvant injection (days)
0.1
0
24
48
72
Fig. 3. Time course of the swelled foot volume of the rats with induced adjuvant
arthritis. Swelled foot volumes are shown as mean ꢀ SE.
Time (hr)
Fig. 5. Plasma concentrations of CS-2100 (8) after oral administration to rats (0.1 and
1 mg/kg) and mice (1 mg/kg). Each value is the mean ꢀ standard deviation of five
animals in the study.
acute increase of the EAE score was observed, reaching a maximal
level on Day 18 (Fig. 4). Mean cumulative EAE scores, calculated by
summing the daily scores, were 14.4, 10.0, 0, and 0 for the vehicle
and 0.1, 0.3 and 1 mg/kg CS-2100 (8) groups, respectively (data not
shown). Compared to the vehicle-treated group, statistically
significant decreases in the cumulative EAE scores were observed
for the 0.3 and 1 mg/kg CS-2100 (8) groups (nonparametric Dun-
nett test [joint ranking], p < 0.01). Thus, CS-2100 (8) has a potent
suppressive effect on EAE in mice, and the above data represent CS-
2100 (8) to be a potential agent for the treatment of MS.
The pharmacokinetics of CS-2100 (8) was evaluated in male
Lewis rats and C57BL/6J mice. The plasma concentration versus
time profiles of CS-2100 (8) in Fig. 5 and the PK parameters of CS-
2100 (8) are presented in Table 2. Mean maximum plasma
concentration (Cmax) at a dose of 1 mg/kg in rats and mice was 186
and 135 ng/ml, respectively, and the time reaching Cmax (Tmax) was
2.0 and 4.0 h post-dose in rats and mice, respectively. In rats, good
dose proportionality was observed for Cmax and AUC0e72 h between
the doses from 0.1 to 1 mg/kg. Terminal-half life (T1/2) in rats and
mice at a dose of 1 mg/kg was 12.4 and 13.9 h, respectively.
While the promising efficacy of CS-2100 (8) as immunosup-
pressant in a variety of preclinical animal disease models was
observed, it was also revealed that CS-2100 (8) had a latent concern
about its metabolic process. From identifying studies on metabo-
lites of CS-2100 (8) in vivo, one of the major metabolites was found
to be 4-Phenoxy benzoic acid (4-PBA, 17) in rat and monkey plasma
after oral administration (Fig. 6), although the preceding in vitro
metabolic stability studies in liver microsomes did not indicate the
generation of 4-PBA (17) as a metabolite nor the degradation of
1,2,4-oxadiazole ring (data not shown). Further detailed analysis
revealed that CS-2100 (8) was subjected to enterobacterial
metabolism in the intestine, resulting in the decomposition of the
1,2,4-oxadiazole ring triggered by the reductive cleavage of the
NeO bond to form 4-PBA (17) (data not shown). This undesirable
ring-opening would arouse concern about potential species or
individual differences in pharmacokinetics and pharmacody-
namics, and accompanying unpredictable toxicological issues. It is
noteworthy that whereas 1,2,4-oxadiazole has been known as an
attractive and versatile heteroaromatic ring in drug design from its
synthetic facility and favorable physicochemical aspect such as
superior oxidative metabolic stability and characteristic moderate
polarity, it has not been publicized enough that 1,2,4-oxadiazoles
have a potential to be subjected to reductive metabolism in liver
and intestinal bacterial flora as described in this report [30e34].
2.0
1.8
1.6
Vehicle
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0.1 mg/kg CS-2100
0.3 mg/kg CS-2100
1 mg/kg CS-2100
3. Conclusion
In summary, we presented CS-2100 (8), a structurally novel S1P1
selective modulator showing potent and selective S1P1 agonist
activity in vitro; peripheral blood lymphocyte lowering and
suppression of the development of adjuvant-induced arthritis in
a rat model and EAE in a mice model. With an unfavorable
Table 2
0
5
10
15
20
25
PK parameters of CS-2100 (8) after oral administration to rats and mice.
Time after EAE induction (days)
Species
Dose
(mg/kg)
Cmax
(ng/ml)
Tmax
(h)
T1/2
(h)
AUC0e72 h
(ng*h/ml)
Fig. 4. Time course of mean EAE scores. The EAE score was evaluated daily from Day 7
to Day 24 in accordance with the following criteria: 0, normal; 1, flaccid tail without
difficulty in picking themselves up; 2, hindlimb weakness defined as paralysis of only
one hindlimb and/or difficulty in picking themselves up; 3, paralysis of both hindlimbs;
4, quadriplesia; 5, dead.
Lewis rats
0.1
1.0
1.0
17.4
186
135
4.0
4.0
2.0
12.4
8.73
13.9
268
2670
1540
C57BL mice

96
T. Nakamura et al. / European Journal of Medicinal Chemistry 51 (2012) 92e98
Fig. 6. 4-Phenoxy benzoic acid resulted from reductive cleavage of the 1,2,4-oxadiazole ring of CS-2100 (8) in rat and monkey.
metabolite 4-PBA (17) and the externally susceptible metabolic
pathway identified, our research effort focused to the substitution
of the central 1,2,4-oxadiazole ring to identify the robust back-up
compound of CS-2100 (8). This continuous study will be reported
elsewhere.
4.1.4. 5-({[tert-Butyl(dimethyl)silyl]oxy}methyl)-3-ethylthiophene-
2-carbonitrile (12)
To a suspension of 11 (1.3 g, 4.8 mmol) and hydroxylamine
hydrochloride (0.37 g, 5.3 mmol) in CH2Cl2 (20 ml) and MeOH
(2 ml) were added triethylamine (1.3 ml, 9.6 mmol) at 25 ꢂC. The
resulting mixture was stirred at 25 ꢂC for 2 h. After removing the
solvent in vacuo, toluene (10 ml) was added to the resulting mixture
and evaporated azeotropically in vacuo. The residue was suspended
in toluene (20 ml) and added dicyclohexylcarbodiimide (1.1 g,
5.3 mmol) at 25 ꢂC. The reaction mixture was stirred at 90 ꢂC for
15 h. After cooling to room temperature, hexane (20 ml) was added
to the reaction mixture, and the resulting mixture was filtered
through Celite. The filtrate was evaporated in vacuo, and the residue
was purified by flash column chromatography (silica gel, hex-
ane:EtOAc 0:10 to 5:95) to afford the title compound 12 (0.94 g,
4. Experimental
4.1. Chemistry
4.1.1. General
NMR spectra were recorded on a Varian Mercury 400 or 500
spectrometer with tetramethylsilane as an internal reference. The
Infrared spectra were recorded on a Jasco FT/IR-830 spectropho-
tometer, and the peaks were recorded in cmꢁ1. The mass spectra
were recorded on a JEOL JMS-AX505H. TLC analysis was performed
on 60F254 plates. Column chromatography was performed on Silica
gel 60 (Merck, 230-400).
3.3 mmol, 69%) as a pale yellow oil. 1H NMR (400 MHz, CDCl3)
d 0.11
(s, 6H), 0.93 (s, 9H), 1.25 (t, 3H, J ¼ 7.8 Hz), 2.75 (q, 2H, J ¼ 7.8 Hz),
4.83 (s, 2H), 6.74 (s, 1H). IR (liquid film): 1093, 1149, 1256,
2212 cmꢁ1. MS (EI): m/z 282 Mþ.
4.1.2. tert-Butyl[(4-ethyl-2-thienyl)methoxy]dimethylsilane (10)
To a solution of [(4-bromo-2-thienyl)methoxy]-tert-butyldime-
thylsilane 9 (0.61 g, 2.0 mmol) and [1,3-bis(diphenylphosphino)
propane]dichloronickel (54 mg, 0.1 mmol) in Et2O (5 ml) was
slowly added ethylmagnesium bromide (1.0 M in THF, 3.0 ml,
3.0 mmol) at 0 ꢂC. The resulting mixture was warmed to 25 ꢂC and
stirred for 1 h, at which time the reaction was quenched with sat.
aq. NH4Cl (5 ml). The resulting biphasic mixture was poured into
water (20 ml) and extracted with Et2O. The combined organic
layers were washed with brine, dried over MgSO4, filtered and
concentrated. The residue was purified by flash column chroma-
tography (silica gel, hexane:EtOAc 0:10 to 5:95) to afford the title
compound 10 (0.44 g, 1.7 mmol, 95%), as a pale yellow oil. 1H NMR
4.1.5. 5-({[tert-Butyl(dimethyl)silyl]oxy}methyl)-3-ethyl-N0-
hydroxythiophene-2-carboximidamide (13)
To a solution of 12 (0.93 g, 3.3 mmol) in EtOH (5 ml) was added
hydroxylamine (40 wt% in water, 0.5 ml, 6.1 mmol), and stirred at
60 ꢂC for 1 h. After removal of the solvent in vacuo, the residue was
diluted with Et2O, poured into water (20 ml) and extracted with
Et2O. The combined organic layers were washed with brine, dried
over MgSO4, filtered and concentrated. The residue was purified by
recrystallization (hexane:AcOEt 6:4) to afford the title compound 13
(0.60 g, 1.9 mmol, 60%) as a white crystalline solid. 1H NMR
(400 MHz, CDCl3)
d
0.11 (s, 6H), 0.93 (s, 9H), 1.20 (t, 3H, J ¼ 7.8 Hz),
2.76 (q, 2H, J ¼ 7.8 Hz), 4.80 (s, 4H), 6.76 (s,1H), 7.10 (br,1H). IR (KBr):
(400 MHz, CDCl3)
d
0.10 (s, 6H), 0.93 (s, 9H), 1.21 (t, 3H, J ¼ 7.4 Hz),
1059, 1590, 1643, 3284, 3357, 3491 cmꢁ1. MS (EI): m/z 315 Mþ.
2.58 (q, 2H, J ¼ 7.4 Hz), 4.82 (s, 2H), 6.77 (s, 1H), 6.81 (s, 1H). IR
(liquid film): 1077, 1131, 1174, 1255, 1463, 1471 cmꢁ1. MS (FAB): m/z
255 (M ꢁ H)þ.
4.1.6. {4-Ethyl-5-[5-(4-phenoxyphenyl)-1,2,4-oxadiazol-3-yl]-2-
thienyl}methanol (15)
To a solution of 4-phenoxybenzoic acid (0.12 g, 0.53 mmol) in
acetonitrile (4 ml) and tetrahydrofuran (2 ml) was added successively
1-hydroxybenzotriazole (74 mg, 0.55 mmol), 1-ethyl-3-(3-dimethyla
minopropyl)carbodiimide hydrochloride (0.11 g, 0.55 mmol) and 13
(0.16 g, 0.50 mmol), and the resulting mixture was stirred at 50 ꢂC for
30min. Aftercoolingtoroomtemperature, thereactionwasquenched
with water (5 ml), and the resulting biphasic mixture was poured into
water (20 ml) and extracted with ethyl acetate. The combined organic
layers were washed successively with 0.1 M hydrochloric acid, sat. aq.
NaHCO3 and brine, and dried over Na2SO4, filtered and concentrated.
The residue was diluted with THF (5 ml) and added tetrabuty-
lammonium fluoride (1.0M inTHF,1.0ml,1.0mmol), and the resulting
mixturewasstirredat50ꢂCfor2h. Aftercoolingtoroomtemperature,
the reaction mixture was poured into water (20 ml) and extracted
with Et2O. The combined organic layers were washed with brine,
dried over Na2SO4, filteredand concentrated. The residuewaspurified
by flash column chromatography (silica gel, hexane:EtOAc 1:3) to
afford the title compound 15 (0.17 g, 0.45 mmol, 89%) as a pale
4.1.3. 5-({[tert-Butyl(dimethyl)silyl]oxy}methyl)-3-ethylthiophene-
2-carboxaldehyde (11)
To a solution of 10 (1.3 g, 5.6 mmol) in THF (10 ml) was slowly
added n-butyllithium (1.6 M in hexane, 4.2 ml, 6.7 mmol) at ꢁ78 ꢂC,
and warmed to 0 ꢂC. After stirring for 30 min at 0 ꢂC, the reaction
mixture was cooled to ꢁ78 ꢂC again and was slowly added N,N-
dimethylformamide (0.86 ml, 11 mmol). After stirring for 30 min
at ꢁ78 ꢂC, the reaction was quenched with sat. aq. NH4Cl (5 ml). The
resulting biphasic mixture was poured into water (20 ml) and
extracted with Et2O. The combined organic layers were washed
with brine, dried over MgSO4, filtered and concentrated. The
residue was purified by flash column chromatography (silica gel,
hexane:EtOAc 0:10 to 5:95) to afford the title compound 11 (1.3 g,
4.6 mmol, 86%) as a pale yellow oil. 1H NMR (400 MHz, CDCl3)
d 0.12
(s, 6H), 0.94 (s, 9H), 1.29 (t, 3H, J ¼ 7.4 Hz), 2.94 (q, 2H, J ¼ 7.4 Hz),
4.86 (s, 2H), 6.84 (s, 1H), 9.99 (s, 1H). IR (liquid film): 1092, 1156,
1225, 1256, 1461, 1659 cmꢁ1. MS (EI): m/z 285 Mþ.

T. Nakamura et al. / European Journal of Medicinal Chemistry 51 (2012) 92e98
97
yellowish crystalline solid. 1H NMR (400 MHz, CDCl3)
d
1.29 (t, 3H,
solution of the compound was orally administered to rats at
a volume of 5 ml/kg. In control rats, vehicle instead of the sus-
pended solution of the compound was orally administered. Blood
was collected from the postcaval vein of the rats under ether
anesthesia at the indicated time in Fig. 2. Then, the collected blood
was placed into a tube containing EDTA. The absolute number of
lymphocytes in the collected blood was counted using a full blood
count analyzer.
J ¼ 7.4 Hz), 1.88 (t, 1H, J ¼ 5.9 Hz), 3.05 (q, 2H, J ¼ 7.4 Hz), 4.83 (d, 2H,
J ¼ 5.9Hz), 6.96(s,1H), 7.04e7.10(m, 4H), 7.20 (t,1H, J ¼ 7.4 Hz), 7.39(t,
2H, J ¼ 7.4 Hz), 8.12 (d, 2H, J ¼ 9.0 Hz). IR (KBr): 1248,1353,1490,1496,
1515, 1588, 1612, 3356 cmꢁ1. MS (FAB): m/z 379 (M þ H)þ.
4.1.7. Methyl 1-({4-ethyl-5-[5-(4-phenoxyphenyl)-1,2,4-oxadiazol-
3-yl]-2-thienyl}methyl)azetidine-3-carboxylate (16)
To a solution of 15 (0.17 g, 0.44 mmol) in CH2Cl2 (6.0 ml) was
successively added carbon tetrabromide (0.19 g, 0.57 mmol) and
triphenylphosphine (0.15 g, 0.57 mmol) at 0 ꢂC and stirred for 1 h.
The reaction mixture was added successively methyl 3-
azetidinecarboxylate hydrochloride (0.10 g, 0.66 mmol) and N,N-
diisopropylethylamine (0.23 ml, 1.3 mmol) at 0 ꢂC, and warmed to
25 ꢂC. After stirring for 2 h, the reaction was quenched with sat. aq.
NaHCO3 (1.0 ml). The resulting biphasic mixture was poured into
water (20 ml) and extracted with ethyl acetate. The extract was
washed with brine, dried over MgSO4, filtered and concentrated.
The residue was purified by flash column chromatography (silica
gel, hexane:EtOAc 1:3 to 1:2) to afford the title compound 16
(0.19 g, 0.40 mmol, 89%) as a pale yellow oil. 1H NMR (400 MHz,
4.2.3. Evaluation of antiarthritic activity
Lewis rats aged 8 weeks were used for the study. Heat-killed
dried Mycobacterium butyricum were ground on an agate mortar
and then suspended in dry-sterilized liquid paraffin to make
a 2 mg/ml suspension. The resulting suspended solution was then
sonicated and used as adjuvant. Arthritis was induced by intra-
dermal injection of the prepared adjuvant (0.05 ml) into the foot
pad of the right hindlimb of rats in the compound-treated group
and in the control group. Rats that were not treated with adjuvant
were separately used as normal control group. The compound was
suspended in 1% (w/v) methyl cellulose #400 solution and orally
administered to rats in the compound-treated group at a volume of
5 ml/kg once daily from the injection day of the adjuvant (Day 0) for
18 successive days. To rats in the control groups 1% (w/v) methyl
cellulose #400 solution alone was similarly administered. The right
foot volume of each rat was measured by customized apparatus for
determination of the volume at the indicated time in Fig. 3. The
mean swelled volume of each group was thus calculated. Percent
inhibition of swelling of the injected foot of treated animals as
compared with that of the control animals was calculated accord-
ing to the following equation:
CDCl3)
d
: 1.27 (t, 3H, J ¼ 7.4 Hz), 3.03 (q, 2H, J ¼ 7.4 Hz), 3.30e3.40
(m, 3H), 3.59e3.67 (m, 2H), 3.71 (s, 3H), 3.78 (s, 2H), 6.84 (s, 1H),
7.04e7.10 (m, 4H), 7.20 (t, 1H, J ¼ 7.4 Hz), 7.39 (t, 2H, J ¼ 7.4 Hz), 8.11
(d, 2H, J ¼ 9.0 Hz). IR (liquid film): 1168, 1200, 1245, 1346, 1489,
1514, 1589, 1613, 1737 cmꢁ1. MS (FAB): m/z 476 (M þ H)þ.
4.1.8. 1-({4-Ethyl-5-[5-(4-phenoxyphenyl)-1,2,4-oxadiazol-3-yl]-2-
thienyl}methyl)azetidine-3-carboxylic acid (8)
To a solution of 16 (0.18 g, 0.39 mmol) in 1,4-dioxane (3 ml) was
added NaOH (1.0 M in water, 1.2 ml, 1.2 mmol), and stirred at 25 ꢂC
Percentage inhibition of swollen foot volume (%) ¼ {1 ꢁ [(swollen
foot volume of animals treated with a compound) ꢁ (foot volume of
normal control animals)]/[(swollen foot volume of control
animals) ꢁ (foot volume of normal control animals)]} ꢃ 100.
for 2 h. The reaction was quenched with acetic acid (68
ml,
1.2 mmol), and the resulting mixture was evaporated in vacuo. To
the residue thus obtained were added successively MeOH (3 ml)
and water (2 ml) with stirring, and the white solid precipitated was
collected by filtration using a Kiriyama funnel, washed with
a mixed solvent of water and methanol (3:7) and dried in vacuo to
afford the title compound 8 (0.15 g, 0.33 mmol, 86%) as a white
4.2.4. Evaluation of the suppressive effect on EAE in mice
Mycobacterium tuberculosis H37 RA (Difco Laboratories)
suspension (8 mg/ml) in incomplete Freund’s adjuvant (Difco
Laboratories) was mixed with myelin oligodendrocyte
glycoprotein35e55 (MOG35e55, Peptide Institute, Inc.) (4 mg/ml
solution) in physiological saline at equal volumes, and the mixture
was emulsified with a sonicator on ice. C57BL/6J mice (female, 6
weeks of age, Japan SLC, Inc.) were immunized by subcutaneous
crystalline solid. 1H NMR (400 MHz, CD3CO2D)
d 1.30 (t, 3H,
J ¼ 7.4 Hz), 3.09 (q, 2H, J ¼ 7.4 Hz), 3.76e3.87 (m, 1H), 4.33e4.45 (m,
2H), 4.45e4.57 (m, 2H), 4.68 (s, 2H), 7.15 (d, 4H, J ¼ 9.0 Hz), 7.24 (t,
1H, J ¼ 7.4 Hz), 7.32 (s, 1H), 7.45 (t, 2H, J ¼ 7.4 Hz), 8.17 (d, 2H,
J ¼ 9.0 Hz). 13C NMR (500 MHz, CD3CO2D)
d 176.3, 175.9, 165.6,
163.2,156.5, 149.1,135.0,134.0,131.3,131.2,126.3,125.9, 121.2,119.2,
119.0, 56.3, 52.9, 33.9, 24.2, 14.7. IR (KBr): 1167, 1249, 1347, 1489,
1517, 1557, 1592, 1613, 3422 cmꢁ1. MS (FAB): m/z 462 (M þ H)þ.
injection of 50
After immunization on Day 0, 200
pertussis (PT, Calbiochem, 1 g/ml solution) in physiological saline
was injected via the tail vein. On Day 2, the same volume of PT
solution (1 g/ml) was injected. Vehicle (1% (w/v) methyl cellulose
ml of emulsion into each of the right and left axillas.
ml of pertussis toxin, Bordetella
m
4.2. Biology
m
#400 solution) or 0.1, 0.3 or 1 mg/kg compound was administered
orally once daily from Day 0 to Day 23. The EAE score was evaluated
daily from Day 7e24 using the following criteria: 0, normal; 1,
flaccid tail; 2, hindlimb weakness; 3, paralysis of both hindlimbs; 4,
quadriplegia; 5, dead (Mendel et al., 1995). The cumulative EAE
score was calculated by summing up daily scores (Days 7e24).
4.2.1. In vitro agonist-evoked GTP
To measure the functional activation of the S1P receptors, an
agonist stimulation of in vitro [35S] GTP
-S binding assay was per-
g-S binding assay
g
formed as follows. The membrane was homogenized from CHO
cells expressing rat S1P1, rat S1P3, human S1P1 or human S1P3,
respectively, with assay buffer (5 mM TriseHCl, pH7.4, 0.25 M
sucrose, 1 mM EDTA, 1 mM EGTA) and centrifuged at 100,000 ꢃ g
4.3. Pharmacokinetics
for 60 min at 4 ꢂC. For in vitro [35S] GTP
g-S binding assay, serial
dilutions of test compound (FTY720-P, CS-0777-P or CS-2100) were
added to aliquots (1e10 g protein/well) of the membrane and
assayed as described in reference [35].
4.3.1. Pharmacokinetics in rats
m
Pharmacokinetics of CS-2100 (8) in male Lewis rats was evalu-
ated using an aliquot of blood collected at each time point in the
blood lymphocyte count evaluation study (section 4.2.2). The blood
was centrifuged to prepare plasma and the plasma concentration
was measured using liquid chromatography-tandem mass spec-
trometry (LCeMS/MS) system after solid phase extraction. The
LCeMS/MS system consisted of a Prominence LC-20A system
4.2.2. Counting of peripheral lymphocytes
Lewis rats (male, 5 weeks of age, Charles River Japan Inc.) were
used. Five rats/group were used. The compound was suspended in
1% (w/v) methyl cellulose #400 solution (vehicle). Suspended

98
T. Nakamura et al. / European Journal of Medicinal Chemistry 51 (2012) 92e98
G.-J. Shei, I.I. Singer, M. Tian, S. West, V. White, J. Xie, R.L. Proia, S. Mandala,
J. Phamacol. Exp. Ther. 309 (2004) 758e768.
(Shimadzu Corp., Japan) and an API 5000 (Applied Biosystems/MDS
SCIEX). The column used was a Capcell pak C18 ACR (column size:
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3.0 mm I.D. ꢃ 150 mm, particle size: 5
mm, Shiseido Co., Japan).
Analysis was performed using WinNonlinÔ (Pharsight, Palo Alto,
CA) and parameters were estimated by noncompartmental analysis
using mean concentration data from five animals for each time
point.
4.3.2. Pharmacokinetics in mice
Male C57BL16J mice were orally dosed with CS-2100 (8) as
a solution in an independent experiment. CS-2100 (8) was solubi-
lized in dimethyl acetamide/25% (w/v) PurebrightÔ (NOF Corp.,
Japan) solution/distilled water (1/8/1, v/v/v). Suspended solution of
the compound was orally administered to mice at a volume of 5 ml/
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Author contributions
T. Nakamura, M. Asano, Y. Sekiguchi, Y. Mizuno, K. Tamaki, and
T. Nishi contributed to the Design, Synthesis of CS-2100; F. Nara,
Y. Kawase, and M. Nagasaki contributed to the in vitro study of
CS-2100, CS-0777-P and FTY720-P; Y. Yabe, D. Nakai, E. Kamiyama,
and Y. Urasaki-Kaneno contributed to the PK study of CS-2100; and
T. Shimozato, H. Doi-Komuro, T. Kagari, W. Tomisato, R. Inoue, H.
Yuita, K. Oguchi-Oshima, and R. Kaneko contributed to the in vivo
study of CS-2100.
Acknowledgments
We thank Dr. Masakazu Tamura and Dr. Kiyoaki Yonesu for
performing the S1P agonist selectivity assays and their helpful
discussions.
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D. Segal, Y. Marantz, D. McCauley, S. Middleton, J. Siu, R.W. Bürli, J. Buys,
M. Horner, K. Salyers, M. Schrag, H.M. Vargas, Y. Xu, M. McElvain, H. Xu, ACS
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