Discovery of S1P agonists with a dihydronaphthalene scaffold

Article abstract of DOI:10.1016/j.bmcl.2011.05.029

Structure-activity relationship of sphingosine-1-phosphate receptor agonists was examined. Cinnamyl derivative 1 was modified to improve S1P ₁ agonistic activity as well as selectivity over S1P₃ agonistic activity. Dihydronaphthalene derivative 10d was identified as a potent S1P₁ receptor agonist with high selectivity against S1P₃ and enhanced efficacy in lowering peripheral lymphocyte counts in mice.

Full text of DOI:10.1016/j.bmcl.2011.05.029

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3885–3889
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of S1P agonists with a dihydronaphthalene scaffold
Haruto Kurata a, , Kensuke Kusumi a, Kazuhiro Otsuki a, Ryo Suzuki a, Masakuni Kurono a, Yuka Takada b,
⇑
Hiroki Shioya b, Takaki Komiya b, Hirotaka Mizuno b, Takeji Ono b, Hiroshi Hagiya b, Masashi Minami b,
Shinji Nakade b, Hiromu Habashita a,
⇑
a Medicinal Chemistry Research Laboratories, Ono Pharmaceutical Co., Ltd, 3-1-1 Sakurai, Shimamoto, Mishima, Osaka 618-8585, Japan
b Exploratory Research Laboratories, Ono Pharmaceutical Co., Ltd, 17-2 Wadai Tsukuba, Ibaragi 300-4247, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Structure–activity relationship of sphingosine-1-phosphate receptor agonists was examined. Cinnamyl
derivative 1 was modified to improve S1P1 agonistic activity as well as selectivity over S1P3 agonistic
activity. Dihydronaphthalene derivative 10d was identified as a potent S1P1 receptor agonist with high
selectivity against S1P3 and enhanced efficacy in lowering peripheral lymphocyte counts in mice.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 2 April 2011
Revised 7 May 2011
Accepted 10 May 2011
Available online 15 May 2011
Keywords:
S1P agonist
Immunomodulator
Dihydronaphthalene
Sphingosine-1-phosphate (S1P) exerts a variety of biological
activities, including vascular maturation and cell survival.1 S1P is
the natural ligand of a specific family of G-protein coupled recep-
tors known as S1P1–5. One significant achievement in the S1P re-
search field was published in 2002 by Lynch and co-workers.
They reported that FTY720 (Fingolimod, Fig. 1), which was devel-
oped as a novel immunomodulator2 and was recently approved
in Russia and the US for the treatment of multiple sclerosis, is
metabolized across species to a monophosphate ester, which can
activate four S1P receptors (S1P1,3–5) to sequester lymphocytes
from circulation to a secondary lymph tissue compartment.3 It
was also reported that S1P1 is essential for lymphocyte recircula-
tion since S1P1 modulates egress from thymus and peripheral lym-
phoid organs.4 On the other hand, S1P3 agonism does not relate to
lymphocyte recirculation but links to bradycardia in rodents.5
Asymptomatic bradycardia was also observed in clinical studies
with FTY720.6 Hence agonists with selectivity for S1P1 over S1P3
are desired. Herein, we report our efforts to identify orally active
S1P1 agonists, selective over S1P3, with good oral bioavailability
in rats.
around 1 to improve S1P1 agonistic activity and selectivity against
S1P3.
The synthesis of the compounds needed for this study is out-
lined in Schemes 1–3. Compounds 5a–5e and 5g–5j were synthe-
sized by reductive alkylation of the corresponding amino acids
(b-alanine, 3-azetidine carboxylic acid or 4-piperidine carboxylic
acid) with aldehydes 4a–4e, 4g–4h and 4j in 28–78% yield (Scheme
1).8 Cinnamyl aldehydes 4a–4e, 4g were derived from the reduc-
tion of the corresponding nitriles 2a, 2c–2f by diisobutylaluminum
hydride (DIBAL-H) followed by treatment with alkyl bromide in
the presence of inorganic base in 4–60% yield. The syntheses of cin-
namyl aldehydes 4h and 3j were described in a previous report.7
Indene and dihydronaphthalene derivatives 10a–10d were pre-
pared from the corresponding phenols 6a and 6b. Compounds 6a
and 6b were alkylated to give compounds 7a and 7b, which in turn
were reduced to the corresponding alcohols 8a and 8b. Treating
compounds 8a and 8b with POCl3 in DMF furnished aldehydes 9a
and 9b via in situ dehydration followed by formylation.9 Amino
acid 10b was obtained in 37% yield from aldehyde 9b. Similarly,
9a was reacted with b-alanine to yield a mixture of 10a and regio-
isomer 10f in 9% yield. Aldehyde 9c was synthesized from tetra-
lone 7b in three steps (Scheme 2).
In a previous report, we described the identification of cinnamyl
derivative 1 as a S1P agonist.7 Oral administration of 1 in mice in-
duced peripheral lymphocyte lowering (PLL) with 50% effective
dose (ED50) of 4.6 mg/kg at 4 h after oral dosing (Fig. 2). Therefore,
we decided to investigate the structure–activity relationship
Dihydrobenzannulene derivative 10e was derived from
benzocycloheptanone derivative 7e, which was prepared by
H3C
NH2
OR
R = H; FTY720 (Fingolimod)
R = PO3H2 (Fingolimod metabolite)
HCl
OH
⇑
Corresponding authors. Tel.: +81 75 961 1151; fax: +81 75 962 9314.
E-mail address: h.kurata@ono.co.jp (H. Kurata).
Figure 1. Structure of FTY720 (Fingolimod) and major metabolite.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.029

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H. Kurata et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3885–3889
Table 1
Ca2+ Assay
EC50 (uM)
Mouse PLL
ED50 (mg/kg)
The influence of linker length on S1P1 and S1P3 agonistic activities
O
H
Compound Structure
Ca2+ assaya
EC50 m)
N
CO2H
p.o. 4 hr
4.6
hS1P1
0.070
hS1P3
3.3
(l
compound 1
hS1P1 hS1P3
O
Figure 2. Structure and pharmacological profiles of cinnamyl derivative 1.
H
N
5a
5b
0.12
0.15
>10
CO2H
O
intramolecular Friedel–Crafts reaction of carboxylic acid 17
(Scheme 3). Carboxylic acid 17 was prepared from 3-hydroxybenz-
aldehyde 13 in seven steps.
The effect of the length of the alkylene linker between the ter-
minal and central phenyl rings of 1 on its in vitro activity was
investigated. Although, 5a showed comparable S1P1 agonistic
activity as 1 and 5b, 5a showed better selectivity over S1P3 than
1 and 5b.
6.7
H
N
CO2H
Agonistic activity was evaluated by measuring intracellular Ca2+ concentration
stimulation in Chinese Hamster Ovary (CHO) cells stably expressing human S1P1 or
S1P3 receptors, respectively.
a
(5d) did not affect activity on S1P1, replacing the 2-methyl group
with a 2-methoxy group (5e) decreased the S1P1 activity by six-fold
(Table 3).
The effect of a methyl group at the b-position of the amine on
the double bond was investigated. Compound 5j demonstrated a
10-fold increase in S1P1 activity compared with the non-
substituted compound 5a (Tables 1 and 3).
Based on the results shown in Table 3, we decided to convert
the cinnamyl moiety into fused ring systems such as indene, dihy-
dronaphthalene and dihydrobenzannulene scaffolds. Whereas,
Next the effect of conformational restriction of b-alanine in 1
was examined. Whereas, azetidine derivative 5h retained in vitro
S1P1 and S1P3 agonistic activities, piperidine derivative 5i exhib-
10
ited decreased in vitro activity on S1P1 (Table 2).
The effect of substituents on the central phenyl ring was investi-
gated. Introducing a methyl group at the 2-position improved
in vitro S1P1 activity by three-fold (5a vs 5g). However, introducing
a methyl group at the 3-position decreased the activity by two-fold.
While the replacement of the 2-methyl group of 5g with chlorine
R'
2
R'
R'
3
b
a
O
O
O
R
R
R
CHO
CN
2a; R = R' = H
CHO
1a; R = R' = H
3a; R = R' = H
1c; R = H, R' = 3-Me
1d; R = H, R' = 2-Cl
1e; R = H, R' = 2-OMe
2c; R = H, R' = 3-Me
2d; R = H, R' = 2-Cl
2e; R = H, R' = 2-OMe
3c; R = H, R' = 3-Me
3d; R = H, R' = 2-Cl
3e; R = H, R' = 2-OMe
1f ; R = CH2Ph, R' = 2-Me
2f ; R = CH2Ph, R' = 2-Me
3f ; R = CH2Ph, R' = 2-Me
3g; R = H, R' = 2-Me
c
R'
2
3
R'
O
e
f
d
O
H
N
HCl
n
n
CO2H
CHO
4a; R' = H, n = 1
5a; R' = H, n = 1
5b; R' = H, n = 2
5c; R' = 3-Me, n = 1
5d; R' = 2-Cl, n = 1
5e; R' = 2-OMe, n = 1
5g; R' = 2-Me, n = 1
4b; R' = H, n = 2
4c; R' = 3-Me, n = 1
4d; R' = 2-Cl, n = 1
4e; R' = 2-OMe, n = 1
4g; R' = 2-Me, n = 1
g
f
O
CO2H
O
m
N
m
CHO
HCl
5h; m = 0
5i ; m = 1
4h
e
d
O
h
O
R
H
N
HCl
CO2H
CHO
5j
3j; R = H
4j; R = (CH2)4Ph
Scheme 1. Reagent: (a) (EtO)2P(O)CH2CN, NaH, THF (for 1c and 1f) or THF/DMF (for 1a, 1d and 1e); (b) DIBAL-H/PhMe, CH2Cl2 (for 2a) or THF (for 2c, 2e and 2f) or THF/CH2Cl2
(for 2d); (c) trifluoroacetic acid (TFA), PhSMe; (d) Ph(CH2)3+nBr, K2CO3, DMF; (e) (i) b-alanine, NaOH (powder), MeOH (for 4a, 4b, 4d and 4e) or MeOH/THF (for 4c, 4g and 4j),
HC(OMe)3 (for 4e and 4j only), (ii) NaBH4; (f) HCl/1,4-dioxane, 1,4-dioxane (for 5a, 5b, 5d and 5e) or 1,4-dioxane/H2O (for 5c, 5g–5i); (g) (i) 3-azetidine carboxylic acid (for 5h)
or 4-piperidine carboxylic acid (for 5i), NaOH (powder), MeOH, THF, (ii) NaBH4; (h) HCl–aq, THF.

H. Kurata et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3885–3889
3887
*
R =
HO
c
O
O
O
a
b
R
R
+
R
n
n
n
n
CHO
O
O
OH
6a; n = 0
6b; n = 1
7a; n = 0
7b; n = 1
8a; n = 0
8b; n = 1
9a; n = 0
9b; n = 1
H
N
H
N
O
d
O
R
R
R
9a
CO2H
CO2H
10f
10a
O
e
R
e
d
f
H
9b
7b
N
HCl
CO2H
10b
O
O
O
g
R
c
R
CHO
OH
11
9c
8c
O
CO2H
d
R
N
HCl
m
10c; m = 0
10d; m = 1
Scheme 2. Reagent: (a) Ph(CH2)4Br, K2CO3, DMF; (b) NaBH4, MeOH, THF; (c) POCl3, DMF (for 8a and 8b) or DMF/CH2Cl2 (for 11); (d) (i) b-alanine (for 10a–10c) or 3-azetidine
carboxylic acid (for 10d), NaOH (powder), MeOH/THF (for 10a and 10b) or MeOH/THF/i-PrOH (for 10c and 10d), HC(OMe)3 (for 10a) or Na2SO4 (for 10c and 10d), (ii) NaBH4;
(e) HCl/1,4-dioxane, 1,4-dioxane, H2O; (f) MeMgBr/Et2O, THF; (g) p-toluenesulfonic acid (p-TsOH) monohydrate, CH2Cl2.
*
R =
OH
b
a
c
HO
CHO
MOMO
CO2Et
MOMO
13
14
15
HO
O
h
g
R
f
e
d
CO2Et
CO2H
16
17
O
O
O
l
R
j
k
i
R
R
CO2H
N
CHO
O
7e
9e
10e
Scheme 3. Reagent: (a) methoxymethyl chloride (MOMCl), K2CO3, acetone; (b) CH2CHMgBr, THF; (c) CH3C(OEt)3, EtCO2H, PhMe; (d) H2, Pd-C, MeOH; (e) HCl–aq, EtOH; (f)
Ph(CH2)4Br, K2CO3, DMF; (g) NaOH–aq, MeOH, THF; (h) (i) (COCl)2, DMF, CH2Cl2, (ii) SnCl4, PhMe; (i) MeMgBr/THF, THF; (j) p-TsOH monohydrate, CH2Cl2; (k) POCl3, DMF,
CH2Cl2; (l) (i) 3-azetidine carboxylic acid, NaOH (powder), MeOH, THF, HC(OMe)3, (ii) NaBH4.

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H. Kurata et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3885–3889
Table 2
Table 4
The influence of conversion to cyclic amino acid on S1P1 and S1P3 agonistic activities
The influence of conversion of cinnamyl moiety to fused ring systems on S1P1 and
S1P3 agonistic activities and in vivo efficacy
Compound
Structure
Ca2+ assaya
EC50 m)
(
l
*
R =
hS1P1
hS1P3
O
O
Compound Structure
Ca2+ assaya
EC50 m)
Mouse PLLb
ED50 (mg/
kg) p.o. 4 h
5h
0.043
2.4
3.3
CO2H
CO2H
(l
N
N
hS1P1
hS1P3
5i
>10
H
N
O
10ac
0.017
0.057
3.2
N.T.
N.T.
R
CO2H
CO2H
a
See Table 1.
O
R
H
10b
7.8
N
O
R
H
N
10c
0.011
>10
0.34
0.16
N.T.
CO2H
CO2H
Table 3
O
R
The influence of substituent on central phenyl ring and double bond towards S1P1 and
S1P3 agonistic activities
10d
0.0029 >10
N
N
O
R
*
R =
10e
1.3
>10
CO2H
Ca2+ assaya
EC50 m)
a
Compound
Structure
See Table 1.
b
(
l
Peripheral Lymphocyte Lowering; Individual data points for dose–titrations
were the average percentage decrease of peripheral blood lymphocyte counts in
n = 5 animals versus control (n = 5) 4 h after oral administration of the test com-
pound (N.T. = not tested).
hS1P1 hS1P3
O
R
c
Including 25% of regioisomer 10f.
H
N
5c
0.28
>10
>10
CO2H
CO2H
CO2H
CO2H
CO2H
O
R
H
N
5g
5d
5e
0.036
0.024
0.23
cyclic amino acid. Dihydronaphthalene derivatives 10c and 10d
showed significant improvement in in vitro S1P1 activity without
any alteration of S1P3 activity. Furthermore, oral administration
of 10c and 10d induced peripheral lymphocyte lowering (PLL) in
mice with a 10-fold lower ED50 as compared to 1 (Table 4).
We examined the pharmacokinetic profiles of representative
compounds 10c and 10d in rats. At a dose of 1 mg/kg, 10d showed
higher oral exposure than 10c (Table 5). Although the clearance
(CLtot) of the two compounds was similar, the half-life (T1/2) of
10d was approximately two times longer than the one of 10c. This
difference might be due to the larger volume of distribution (Vss) of
10d.
O
R
Cl
H
N
7.2
O
R
OMe
H
N
>10
O
R
H
N
5j
0.010
3.0
a
See Table 1.
In summary, we explored detailed structure–activity relation-
ships (SAR) of cinnamyl derivative 1 to identify dihydronaphtha-
lene derivative 10d, which showed potent hS1P1 agonistic
activity with excellent selectivity over hS1P3 and in vivo PLL
activity in mice. It is noteworthy, that the dihydronaphthalene
scaffold can be found in a limited number of biologically active li-
gands, including prostaglandin I2 receptor (IP) agonists,11a throm-
boxane A2 synthase inhibitors,11b NMDA receptor blockers,11c
dihydrobenzannulene 10e showed modest S1P1 activity, both in-
dene 10a and dihydronaphthalene 10b demonstrated improved
S1P1 activity without modifying S1P3 activity (Table 4). Since in-
dene derivatives have potential to isomerize, no further investiga-
tion on this scaffold was performed. Next, we investigated the
introduction of a methyl group to the 1-position of the 3,4-
dihydronaphthalene scaffold and the conversion of b-alanine to a
Table 5
a
Pharmacokinetic profiles of 10c and 10d in rat (1 mg/kg)
Compound
AUCinf
5.4
( 0.47)
2.4
( 0.37)
4.4
( 1.2)
3.8
(
lg h/mL)
Cmax (ng/mL)
CLtot (mL/min/kg)
T1/2 (h)
Vss (L/kg)
BA (%)
44
10c
i.v.
—
3.1
( 0.3)
—
5.2
( 0.3)
6.9
( 1.5)
11.0
( 3.5)
16.7
0.86
( 0.13)
—
p.o.
i.v.
168
( 23)
—
10d
4.0
( 1.0)
—
2.86
( 0.46)
—
85
p.o.
181
( 0.30)
( 33)
( 9.0)
a
Values are means of three experiments, standard deviation is given in parentheses.

H. Kurata et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3885–3889
3889
5-HT1A/D-2 antagonists,11d human leukocyte elastase inhibitors11e
and PPAR modulators.11f Further optimization of compound 10d
will be reported in due course.
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Acknowledgments
Authors thank Mr. T. Shono and Mr. N. Matsumura for their con-
tribution to measuring the PK profile and acknowledge Dr. I.C.
Choong for preparation of this manuscript.
8. Verardo, G.; Geatti, P.; Pol, E.; Giumanini, A. G. Can. J. Chem. 2002, 80, 779.
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