Discovery of CS-2100, a potent, orally active and S1P3-sparing S1P1 agonist

Article abstract of DOI:10.1016/j.bmcl.2011.12.019

S1P₃-sparing S1P₁ agonists have attracted attention as a suppressant of autoimmunity with reduced side effects. Our synthetic efforts and extensive SAR studies led to the discovery of 10b named CS-2100 with the EC₅₀ value of 4.0 nM for human S1P₁ and over 5000-fold selectivity against S1P₃. The in vivo immunosuppressive efficacy was evaluated in rats on host versus graft reaction and the ID ₅₀ value was determined at 0.407 mg/kg. The docking studies of CS-2100 with the homology model of S1P₁ and S1P₃ showed that the ethyl group on the thiophene ring of CS-2100 was sterically hindered by Phe263 in S1P₃, not in the case of Leu276 in S1P₁. This observation gives an explanation for the excellent S1P₃-sparing characteristic of CS-2100.

Full text of DOI:10.1016/j.bmcl.2011.12.019

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1788–1792
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery 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,
Takako Kimura b, Futoshi Nara c, Yumi Kawase c, Takaichi Shimozato d, Hiromi Doi d, Takashi Kagari d,
Wataru Tomisato d, Ryotaku Inoue d, Miyuki Nagasaki d, Hiroshi Yuita e, Keiko Oguchi-Oshima e,
Reina Kaneko f, Nobuaki Watanabe g, Yasuyuki Abe h, 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 Lead Discovery & Optimization Research Laboratories II, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
c Cardiovascular Metabolics Research Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
d Frontier Research Laboratories, Daiichi Sankyo Co., Ltd, 1-16-13 Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan
e Oncology Research Laboratories, Daiichi Sankyo Co., Ltd, 1-16-13 Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan
f Global Project Management Department, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
g Drug Metabolism & Pharmacokinetics Research Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
h Medicinal Safety Research Laboratories, Daiichi Sankyo Co., Ltd, 1-16-13 Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
S1P3-sparing S1P1 agonists have attracted attention as a suppressant of autoimmunity with reduced side
effects. Our synthetic efforts and extensive SAR studies led to the discovery of 10b named CS-2100 with
the EC50 value of 4.0 nM for human S1P1 and over 5000-fold selectivity against S1P3. The in vivo immu-
nosuppressive efficacy was evaluated in rats on host versus graft reaction and the ID50 value was deter-
mined at 0.407 mg/kg. The docking studies of CS-2100 with the homology model of S1P1 and S1P3
showed that the ethyl group on the thiophene ring of CS-2100 was sterically hindered by Phe263 in
S1P3, not in the case of Leu276 in S1P1. This observation gives an explanation for the excellent S1P3-spar-
ing characteristic of CS-2100.
Received 29 October 2011
Revised 25 November 2011
Accepted 3 December 2011
Available online 2 January 2012
Keywords:
S1P1
Agonist
CS-2100
Lymphocyte
HvGR
Ó 2011 Elsevier Ltd. All rights reserved.
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 as a sup-
pressant of autoimmunity by affecting lymphocyte trafficking,
through the 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)3, which is an agonist of
S1P1,3,4,5 but not of S1P2. From intense studies on both FTY720 and
S1P receptors, it has also been 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 Re-
cent studies suggested that only removal of the S1P3 agonism is
insufficient to exclude the cardiovascular side effect.6a However, a
great deal of research efforts focused on the exploration of S1P3-
sparing S1P1 agonists,6 with an aim to reduce potential side effects
but preserve the impressive efficacy as demonstrated in clinical tri-
als of FTY720 for the treatment of transplant rejection7 and remit-
ting relapsing multiple sclerosis.8
NH
2OH
OR
NH2
CH3(CH2)12
O
O OH
OH
P
CH3(CH2)7
OH
2
, R = H, FTY720
1
(S1P)
3
, R = PO3H2, FTY720-P
O N
N
CO2H
O N
N
CF3
N
S
S
F3C
F3C
4
5
(Merck)
(SEW2871)
⇑
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, FTY720, FTY720-P, SEW2871 and one of the 1,2,4-
oxadiazole derivatives reported by Merck.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.12.019

T. Nakamura et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1788–1792
1789
In 2004, Scripps and Norvartis reported SEW2871 (4),5b,9 a non
a - c
S
S
OTBS
OTBS
phosphate type S1P1 selective agonist, which has attracted atten-
tion as the second stream of S1P1 agonists. On the other hand,
Merck also disclosed a series of non phosphate type S1P1 agonists,
representatively, compound 5 in their precedent patent.10 Encour-
aged by our success in the discovery of a phosphate type S1P1 ago-
nist, CS-0777 as a promising clinical candidate,11 we continued to
investigate new S1P1 agonists and initiated the SAR studies based
on the structure of compound 5. Herein, we describe the discovery
process for a potent S1P3-sparing S1P1 agonist, CS-2100 and report
its SAR study with consideration of the homology model based on
the X-ray crystal structure of bovine rhodopsin.
Br
CHO
OH
NC
NC
6a
11
d - g
S
S
12
6b
6c
Based on the structure of 5, we planned to (1) convert the inner
benzene ring to other aromatics and (2) transform the left-hand
side 4-phenyl-5-trifluoromethyl thiophene structure to procure a
patentable basal structure.
b, c
OH
OTBS
Br
NC
S
S
13
The general synthetic method is outlined in Scheme 1. Aromatic
nitriles 6 were reacted with aqueous hydroxyamine to give ami-
doximes 7. Subsequent 1,2,4-oxadiazole formation12 provided a
series of 1,2,4-oxadiazoles 8 with simultaneous deprotection of
the TBS group except for the THP-protected one. As for THP-depro-
tection, it was conducted after 1,2,4-oxadiazole formation. Bromin-
ation or chlorination of 8 and the succedent substitution by methyl
azetidine-3-carboxylate, followed by hydrolysis provided 9a–m
and 10a–r.
Scheme 2. Reagents and conditions: (a) NaBH4, MeOH (quant.); (b) TBSCl,
imidazole, DMF (quant.); (c) CuCN, DMF (58% for 6a, 56% for 6c); (d) TBSCl,
imidazole, DMF (86%). (e) n-BuLi, THF, then DMF (54%); (f) NH2OH–HCl, Et3N,
CH2Cl2–MeOH; (g) DCC, toluene, 90 °C (95%, two steps).
Me
Me
Me
NC
a, b
c - e
OTBS
OTBS
OTBS
CO2H
CHO
S
S
S
S
Regarding the preparation of aromatic nitriles 6, the synthetic
routes are summarized in Scheme 2. 6a was prepared from aldehyde
11 by reduction, TBS-protection and following cyanidation with
copper cyanide. 6b was obtained from alcohol 12 by TBS-protection,
formylation and nitrile formation through dehydration of the oxime.
6c was provided by the similar method for preparation of 6a.
For the purpose of structural optimization, our synthetic effort
was extended to various alkylated thiophenes. The synthetic routes
are summarized in Scheme 3. Starting from carboxylic acid 14,
reduction and TBS-protection provided 15a. Subsequent formyla-
tion, oxime formation and dehydration gave 6d. Regarding 6e–g,
aldehyde 11 was converted to 15b–d, respectively by reduction,
TBS-protection and a Ni-catalyzed cross coupling reaction with
the corresponding Grignard reagents. As for 15d, isopropenyl mag-
nesium bromide was used for a cross-coupling reaction and then
the olefin was hydrogenated because the use of isopropyl magne-
sium bromide resulted in a mixture of n-propylated and isopropy-
lated compounds. The transformation of 15b–d to 6e–g was
performed by the same method as the conversion from 15a into
6d. Compound 6h was prepared from carboxylic acid 16 in the
same manner as preparation of 6d. 6i, which is the only THP-pro-
tected thiophene, was synthesized via the intermediate 17 pre-
pared from 15b in four steps including formylation, reduction,
14
11
15a
6d
Br
R
R
f - h
c - e
OTBS
NC
S
S
15b R = Et
6e R = Et
15c R = n-Pr
6f R = n-Pr
15d
R = i-Pr
6g
R = i-Pr
Me
Me
Me
a, b
c - e
OTBS
OTBS
CO2H
16
NC
NC
S
S
S
15e
6h
Et
S
Et
Et
i - l
m - o
TBSO
HO
OTHP
OTHP
S
S
15b
17
6i
Scheme 3. Reagents and conditions: (a) BH3, THF; (b) TBSCl, imidazole, DMF (67%
for 15a, 88% for 15e, two steps); (c) n-BuLi, DMF, THF (47–89%); (d) NH2OH–HCl,
Et3N, CH2Cl2–MeOH; (e) DCC, toluene, 90 °C (69–100%, two steps); (f) NaBH4,
MeOH; (g) TBSCl, imidazole, DMF (quant. two steps); (h) EtMgBr, NiCl2(dppp), THF
(95%) for 15b. n-PrMgBr, NiCl2(dppp), Et2O (72%) for 15c. Isopropenyl magnesium
bromide, NiCl2(dppp), Et2O (82%), then H2, RhCl(PPh3)3, benzene (86%) for 15d; (i) n-
BuLi, DMF, THF (86%); (j) NaBH4, MeOH (94%); (k) 3,4-dihydro-2H-pyrane, p-TsOH;
(l) TBAF, THF (78%, two steps); (m) PDC, MS4A, CH2Cl2 (87%); (n) NH2OH–HCl, Et3N,
CH2Cl2; (o) DCC, toluene, 90 °C (95%, two steps)
a
N
Ar OPG
NH2
b or c, (d)
HO
Ar OPG
NC
PG = TBS or THP
6
7
F
O
OH
1-3
a, b
1-3
R
+
R
e - g
O N
N
O N
CHO
CO2H
R4, 5
CO2H
R4, 5
R
Ar
R
Ar
N
N
OH
18
19
20
8
9a-m, 10a-r
Scheme 4. Reagents and conditions: (a) K2CO3, DMF, 100 °C; (b) NaClO2, KH2PO4, 2-
methyl-2-butene, THF-t-BuOH–H2O (33%-quant., two steps)
Scheme 1. Reagents and conditions: (a) aq. NH2OH, EtOH (52–98%); (b) RCOCl,
Et3N, CH2Cl2, then TBAF, THF (65–98%); (c) RCO2H, WSC–HCl or DCC, HOBt, CH3CN,
then TBAF, THF (40-94%); (d) PPTS, EtOH, 60 °C (92%) for THP deprotection; (e) CBr4,
PPh3, CH2Cl2 or SOCl2, cat. DMF, toluene; (f) Methyl azetidine-3-carboxylate-HCl or
Ethyl azetidine-3-carboxylate-HCl, i-Pr2NEt, CH3CN (44–93%, two steps). (g) 1 N aq.
NaOH or LiOH, then AcOH or oxalic acid (10c, 10g, 10h and 10i were solidified as
oxalates.), (39–95%).
THP-protection and TBS-deprotection. Subsequent oxidation by
PDC and following nitrile formation through dehydration of the
oxime provided 6i.

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T. Nakamura et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1788–1792
Table 1
Table 3
SAR of 1,2,4-oxadiazole derivativesa
SAR of alkyl thiophene derivativesa
R6
O N
N
O
R7
N
Ar
CO2H
R
N
N
N
O
S
CO2H
Compound
R
Ar
c
-GTP EC50 (nM)
Ratio
hS1P1
hS1P3
Compound
R6
R7
c
-GTP EC50 (nM)
Ratio
hS1P1
hS1P3
9h
H
Me
Et
n-Pr
i-Pr
H
H
H
H
H
H
Me
Et
1.0
11
4.0
30
75
25
12
900
900
5
4.2
1600
380
123
S
S
10a
10b
10c
10d
10e
10f
>20000
>20000
13000
15000
>20000
>20000
>1818
>5000
433
200
>800
>1666
F3C
F3C
S
9a
6.5
800
H
S
S
S
9b
9c
9d
850
>20000
>20000
>20000
>23
>666
1
a
EC50 is defined as the midpoint between the binding or inhibitory ratio of the
vehicle and the maximum response of the test compound.
30
(EC50 = 8.5 nM) but also superior S1P1/S1P3 selectivity to 5 (541-
fold). In addition, comparing analogues which differ in the position
of the sulfur atom in the central thiophene ring (9f–9h), 9h also
showed satisfactory S1P1 agonist activity (EC50 = 1 nM) and favor-
able S1P1/S1P3 selectivity (900-fold).
The identification of both 9f and 9h prompted us to investigate
further SAR studies aiming to improve their mediocre S1P1/S1P3
selectivity, which were started from introducing a methyl group
to various structural positions of 9f and 9h.
The result of methyl-introduced derivatives is shown in Table 2.
Regarding the derivatives of 9f, introduction of a methyl group to
the R1 (9i), R2 (9j) or R5 (9m) position worsened S1P1 agonist activ-
ity considerably. Aside from the above three, R3 (9k) and R4 (9l)
positions exhibited acceptable tendencies (compared 9f, 9k and
9l). On the other hand, based on 9h, drastic improvement in
S1P1/S1P3 selectivity was observed by introduction of a methyl
group to R6 on the thiophene ring (10a; >1818-fold).
Therefore, we turned our attention to introducing substituent
on the thiophene ring of 9h. The result of the introduction of var-
ious alkyl groups on the thiophene ring of 9h is summarized in Ta-
ble 3. Introduction of the ethyl group (10b) increased S1P1/S1P3
selectivity outstandingly, showing over 5000-fold with preserving
favorable S1P1 agonist activity (EC50 = 4.0 nM). Meanwhile, n-Pr
(10c) and i-Pr (10d) worsened not only S1P1/S1P3 selectivity but
also in vitro activities. On the other hand, the introduction of alkyl
groups in the R7 position such as methyl (10e) and ethyl (10f) also
influenced S1P1/S1P3 selectivity. Especially in the case of ethyl
(10f), increased S1P1/S1P3 selectivity was observed. However, both
derivatives decreased S1P1 agonist activity after all.
>20000
O
S
S
9e
9f
60
>20000
4600
>333
541
O
O
O
O
8.5
34
1.0
S
9g
9h
>20000
900
>588
900
S
a
EC50 is defined as the midpoint between the binding or inhibitory ratio of the
vehicle and the maximum response of the test compound.
For further scrutinization, a series of benzoic acids 20 were pre-
pared as shown in Scheme 4, by SNAr reaction of phenols 1813 with
4-fluorobenzaldehydes 19, followed by Pinnick oxidation.
The agonist activities against human S1P1 and S1P3 were mea-
sured by [35S]GTP
c-S binding assay. The SAR information about
the inner aromatic ring (Ar) and the left-hand side structure (R)
is summarized in Table 1. It was revealed that the replacement
of the inner benzene ring to thiophene was accepted and showed
fair S1P1 affinity, although S1P1/S1P3 selectivity had deteriorated
(compare 5 with 9a). Encouragingly, from the result of the trans-
formation of the left-hand side structure into biphenyl (9b, 9c),
benzyloxyphenyl (9d), alkyloxy phenyl (9e) or phenoxy phenyl
(9f), lattermost 9f displayed not only good S1P1 agonist activity
Table 2
SAR of methyl-introduced derivativesa
R1
R2
R6
X
O N
N
O
Y
R3
R5
CO2H
N
R4
Compound
X
Y
R1
R2
R3
R4
R5
R6
c
-GTP EC50 (nM)
Ratio
hS1P1
hS1P3
9f
9i
9j
9k
9l
9m
9h
10a
S
S
S
S
S
S
CH
CH
CH
CH
CH
CH
CH
CH
S
H
Me
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
Me
8.5
110
200
35
4600
541
109
>100
57
142
>33
900
>1818
12000
>20000
2000
3000
>20000
900
21
600
1.0
11
S
H
H
>20000
a
EC50 is defined as the midpoint between the binding or inhibitory ratio of the vehicle and the maximum response of the test compound.

T. Nakamura et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1788–1792
1791
Table 4
Scrutinization of substituents on phenoxy phenyl structurea
O N
N
O
S
R3
CO2H
N
R4
Compound
R3
R4
c
-GTP EC50 (nM)
Rat HvGRb ID50 (mg/kg)
or % inhibition @ 1 mg/kg
hS1P1
hS1P3
10b
10g
10h
10i
10j
10k
10l
10m
10n
10o
10p
10q
10r
H
F
Cl
MeO
Me
Et
n-Pr
n-Bu
i-Pr
H
H
H
H
H
H
H
H
H
H
F
Cl
MeO
Me
4.0
8.5
6.5
4.0
3.0
6.5
13
9.0
4.5
5.0
5.0
14
>20000
>20000
>20000
15000
4500
10000
>20000
>20000
11000
>20000
>20000
>20000
6000
0.407
0.369
0.461
74%
35%
62%
56%
66%
NTc
0.142
49%
H
H
H
NTc
7.5
NTc
a
b
c
EC50 or ID50 is defined as the midpoint between the binding or inhibitory ratio of the vehicle and the maximum response of the test compound.
Orally-administered once daily for four successive days from the day of immunization.
Not tested.
Subsequently, we focused on further scrutinization of the sub-
stituents on phenoxy phenyl structure of 10b, in light of the al-
ready-obtained SAR information shown in Table 2 that R3 and R4
positions displayed acceptable tendencies. In addition to in vitro
agonist activities, regarding the in vivo immunosuppressive effect
on transplant rejection, we evaluated immunosuppressive efficacy
in rats on host versus graft reaction (HvGR).14
The result of ethyl thiophene derivatives variously-functional-
ized on the phenoxy phenyl group of 10b is summarized in Table 4.
In R3 substituent, introduction of the fluorine (10g), chlorine
(10h), methoxy (10i) or methyl (10j) group showed good S1P1
agonist activity (EC50 = 3.0 to 8.5 nM). In particular, 10g and
10h exhibited favorable S1P1/S1P3 selectivity and inhibitory
activities in rats on HvGR (ID50 = 0.369 and 0.461 mg/kg, respec-
tively) on par with 10b (ID50 = 0.407 mg/kg). Interestingly, good
S1P1 agonist activities were also observed in even bulky substi-
tuent such as ethyl (10k), n-propyl (10l), n-butyl (10m) or iso-
propyl (10n) (EC50 = 4.5 to 13 nM). On the other hand, in R4
substituent, fluoro (10o), chloro (10p), methoxy (10q) and
methyl (10r) derivatives showed fair S1P1 agonist activity as
well (EC50 = 5.0 to 14 nM). Besides this, 10o and 10p showed
good S1P1/S1P3 selectivity and 10o exhibited distinguished
in vivo efficacy (ID50 = 0.142 mg/kg).
Based on examinations of 10b, 10g, 10h and 10o to evaluate
their toxicological aspects, physicochemical profiles, including
crystallinity, hygroscopicity, and physical stability (date not
shown), we selected 10b as a clinical candidate named CS-2100.
The success in the discovery of CS-2100 was greatly contributed
to by the dramatic improvement of S1P1/S1P3 selectivity resulting
from introduction of the ethyl group in R6 position of the thio-
phene ring. This experimental result led us to evaluate the receptor
binding models. The homology model of S1P1 was built based on
the X-ray crystal structure of bovine rhodopsin. Meanwhile, the
S1P3 was modeled based on the S1P1 homology model in order
to focus on the nonconserved residues.
Figure 2. Docking model of 10b (CS-2100) into the S1P1 homology model. The S1P3
Phe263 residue (orange) interferes with the ethyl group of 10b.
Interestingly, the ethyl group on the thiophene ring suffered
from severe steric hindrance caused by Phe263 in S1P3, not in
the case of Leu276 in S1P1. This observation gives an explanation
for the excellent S1P3-sparing characteristic of CS-2100. From re-
cent information, the similar contribution by the Leu276/Phe263
residues for S1P1/S1P3 selectivity has also been reported in other
S1P1 agonists.15b,c
In addition, it was found that Val132, Leu128, Phe205, Val209,
Phe265 and Leu213 formed a hydrophobic subpocket in S1P1, as
shown in Figure 3. Our further docking studies indicated that
rather hydrophobic substituents such as the n-Pr or n-Bu group
in R3 position (10l or 10m, Table 4) are accommodated by this sub-
pocket to maintain good S1P1 affinity. This observed tolerance for
introducing relatively larger functional groups would afford en-
ough room to modulate physicochemical properties without loss
of biological activities in this class of compounds.
The docking model of 10b (CS-2100) into S1P1 homology model
is illustrated in Figure 2. In our model, the charged azetidine car-
boxylic acid structure favorably interacted with Glu121 and
Arg120 of S1P1. This interaction is considered to be important for
recognition of S1P receptor agonists or natural ligand S1P as re-
ported by Parrill et al.15a
In summary, we have reported the discovery of CS-2100, a po-
tent, orally active, S1P3-sparing S1P1 agonist. 4-Ethyl thiophene
structure of CS-2100 is profitable for the S1P3-sparing property,

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Figure 3. Hydrophobic subpocket in the docking model of 10m into S1P1 homology
model. n-Bu group of 10m is placed in the subpocket.
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suggested by the result of docking studies with homology models
as well as SAR studies.
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Neway III, W. E. WO03062252, 2003.; (b) Hale, J. J.; Lynch, C. L.; Neway, W.;
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P+Ph3Br-
CHO
OH
H2, Pd-C
t-BuOK
OH
OH
EtOH
DMF
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(quant.)
(quant.)
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