Structure-activity relationship studies of S1P agonists with a dihydronaphthalene scaffold

Article abstract of DOI:10.1016/j.bmcl.2011.11.048

Structure-activity relationship (SAR) of sphingosine-1-phosphate receptor agonists with a dihydronaphthalene scaffold was investigated. Compound 1 was modified to improve S1P₁ agonistic activity and in vivo peripheral lymphocyte lowering (PLL) activity without impairing selectivity over S1P ₃ agonistic activity. A detailed SAR study of the terminal lipophilic part revealed that the introduction of substituents on the propylene linker and the terminal benzene ring influences in vitro and PLL activities. Compound 6n bearing a (S)-methyl group at the 2-position on the propylene linker and chlorine at the para-position on the terminal benzene ring showed potent hS1P₁ agonistic activity with excellent selectivity over hS1P ₃ and in vivo PLL activity in mice.

Full text of DOI:10.1016/j.bmcl.2011.11.048

Bioorganic & Medicinal Chemistry Letters 22 (2012) 144–148
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationship studies of S1P agonists with a
dihydronaphthalene scaffold
Haruto Kurata a, , Kensuke Kusumi a, Kazuhiro Otsuki a, Ryo Suzuki a, Masakuni Kurono a,
⇑
Natsuko Tokuda a, Yuka Takada b, Hiroki Shioya b, Hirotaka Mizuno b, Takaki Komiya 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 (SAR) of sphingosine-1-phosphate receptor agonists with a dihydronaph-
thalene scaffold was investigated. Compound 1 was modified to improve S1P1 agonistic activity and
in vivo peripheral lymphocyte lowering (PLL) activity without impairing selectivity over S1P3 agonistic
activity. A detailed SAR study of the terminal lipophilic part revealed that the introduction of substituents
on the propylene linker and the terminal benzene ring influences in vitro and PLL activities. Compound
6n bearing a (S)-methyl group at the 2-position on the propylene linker and chlorine at the para-position
on the terminal benzene ring showed potent hS1P1 agonistic activity with excellent selectivity over hS1P3
and in vivo PLL activity in mice.
Received 21 September 2011
Revised 10 November 2011
Accepted 14 November 2011
Available online 20 November 2011
Keywords:
S1P agonist
Immunomodulator
Dihydronaphthalene
Ó 2011 Elsevier Ltd. All rights reserved.
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. A 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 several countries 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, in rodents, S1P3 agonism is not
related to lymphocyte recirculation but instead is linked to brady-
cardia.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 ac-
tive S1P1 agonists which are selective over S1P3 with good oral bio-
availability in rats.
lowering (PLL) with a 50% effective dose (ED50) of 0.16 mg/kg at
4 h and 1.9 mg/kg at 24 h after oral dosing (Fig. 2).8 Therefore,
we decided to investigate the structure–activity relationship
around 1 to improve the S1P1 agonistic activity and in vivo efficacy
without impairing selectivity over S1P3.
Compounds were synthesized as shown in Schemes 1–4. Com-
pound 6a was prepared by a linear synthetic procedure as de-
scribed in a previous report.7 6-Hydroxytetralone 2 was treated
with 3-phenylpropyl bromide in the presence of potassium car-
bonate in DMF to yield 6-(3-phenylpropyloxy)tetralone 3a in 94%
yield. The methyl group was incorporated using a methyl Grignard
reagent to the ketone 3a to yield tetrahydronaphthalenole 4. Treat-
H3C
NH2
OR
R = H; FTY720 (Fingolimod)
R = PO3H2 (Fingolimod metabolite)
HCl
OH
Figure 1. Structure of FTY720 (Fingolimod) and major metabolite.
In a previous report,7 we described the identification of dihy-
dronaphthalene derivative 1 as a potential scaffold with good
S1P1 agonist activity, selectivity over S1P3 and in vivo efficacy. Oral
Ca2+ Assay
EC50 (nM)
Mouse PLL
ED50 (mg/kg)
O
CO2H
N
p.o. 24 hr
1.9
p.o. 4 hr
0.16
hS1P1
2.9
hS1P3
administration of
1 in mice induced peripheral lymphocyte
>10000
compound 1
Figure 2. Structureandpharmacologicalprofilesofdihydronaphthalenederivative1.
⇑
Corresponding authors. Tel.: +81 75 961 1151; fax: +81 75 962 9314.
E-mail address: h.kurata@ono.co.jp (H. Kurata).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.048

H. Kurata et al. / Bioorg. Med. Chem. Lett. 22 (2012) 144–148
145
O
HO
a
R
R =
HO
*
CHO
CHO
O
O
a
b
5c-5n
8
R
R
OH
c
e
b
d
O
O
O
5c-5i
5j-5n
4
CO2H
R
3a
2
N
(HCl)
O
O
e
CO2H
d
c
R
6c-6n
R
N
CHO
HCl
f
6a
5a
CO2H
CO2Me
HCl
HN
9
HN
Scheme 1. Reagents: (a) Ph(CH2)3Br, K2CO3, DMF; (b) MeMgBr/Et2O, THF; (c) POCl3,
DMF, CH2Cl2; (d) (i) 3-azetidine carboxylic acid, NaOH (powder), HC(OMe)3, MeOH,
THF; (ii) NaBH4; (e) HCl-aq, THF.
10
Scheme 3. Synthesis of 6c–6n. Reagents: (a) the corresponding alcohol 12c–12n,
DEAD (for 12c and 12e) or TMAD (for 12d and 12f–12n), PPh3, THF; (b) 3-azetidine
carboxylic acid 9, NaOH (powder), MeOH, THF, HC(OMe)3; (ii) NaBH4; (c) HCl-aq,
THF; (d) 3-azetidine carboxylic acid methyl ester 10, NaBH(OAc)3, Et3N/THF (for 5j
and 5n) or AcOH/DMF (for 5k–5m); (e) NaOH-aq, MeOH; (f) SOCl2, MeOH.
ment of alcohol 4 with Vilsmeier reagent generated by POCl3 in
DMF yielded dihydronaphthalene-2-carboaldehyde 5a via dehy-
dration in situ in 42% yield from 3a.9 Aldehyde 5a was treated with
3-azetidine carboxylic acid 9 in the presence of NaOH powder and
methyl orthoformate. The resulting iminium salt was reduced
in situ by sodium borohydride to yield the amino acid,10 which
was subsequently treated with HCl-aq to be prepared as the HCl
salt 6a in 46% yield (Scheme 1).
Compounds 6c–6n were prepared by a convergent synthetic
procedure using phenol-aldehyde 8 as a key intermediate. First,
intermediate 8 was prepared by modifying the method of Scheme
1. 6-Benzyloxy tetralone 3b was obtained by a similar procedure to
3a in 87% yield. The methyl group at 1-position of dihydronaphtha-
lene was incorporated by addition of a methyl Grignard reagent
followed by dehydration with aqueous HCl to yield 6-benzyloxy
dihydronaphthalene 7 in 38% yield. The benzyl group of compound
5b, which was obtained in 62% yield from 7 by a similar procedure
to aldehyde 5a, was removed in TFA in the presence of thioanisole
to yield the key intermediate 8 in 59% yield (Scheme 2).
The phenylpropyl moiety was incorporated into the phenol part
of the key intermediate 8 by Mitsunobu reaction with the corre-
sponding alcohol 12c–12n to yield aldehydes 5c–5n in 27% to
quantitative yield. Finally, the amino acid moiety was incorporated
by two procedures. Compounds 6c–6i were obtained from 5c–5i
by a similar procedure to 6a in 34–58% yield. Compounds 5j–5n
were treated with 3-azetidine carboxylic acid methyl ester hydro-
chloride 10 in the presence of sodium triacetoxyborohydride to
yield the corresponding 3-azetidine carboxylates in 37–94% yield,
which were converted by saponification to amino acids 6j–6n in
42–89% yield (Scheme 3).
prepared as shown in Scheme 4. 3-Phenylpropanoic acids 11c–
11g were treated with a BH3 THF complex to yield alcohols 12c–
12g in 57% to quantitative yield. Ethyl ester 13 was treated with
LiAlH4 to yield alcohols 12i in 94% yield. Evans’ chiral oxazolidinone
auxiliary11 15S was treated with n-BuLi followed by acid chloride
14j to yield 30-acyloxazolidinone 16j (40-S) in quantitative yield,
which was treated with sodium hexamethyldisilazane (NaHMDS)
followed by p-fluorobenzyl bromide to yield 2-benzylated-30-acy-
loxazolidinone 17j (2-R, 40-S) in 56% yield as a single diastereomer.
Compound 17j was treated with LiBH4 to yield alcohol 12j as an
optically pure form in 86% yield. Other alcohols 12k–12n were also
prepared by a similar procedure to 12j (Scheme 4).
First, the effect of introducing a fluorine substituent on the ter-
minal phenyl ring of 6a was investigated through the in vitro and
in vivo activity. While introducing fluorine at the meta (6d) or ortho
(6e) position on the terminal phenyl ring showed comparable
in vitro activity, 6c bearing a fluorine atom at the para-position
showed an improved S1P1 agonistic activity. Then, the in vivo
PLL activity of 6c–6e was examined. At 4 h after oral administra-
tion, compounds 6c–6e showed improved activity by 3- to 7-fold
compared to 6a and at 24 h after oral administration they showed
3- to 8-fold better activity than 1 (Table 1).
Next, the effect of a methyl group on the alkylene linker be-
tween the aromatic rings of 6a was investigated. Whereas 6h bear-
ing a methyl group at the 1-position of the alkylene linker showed
modest S1P1 activity, 6f or 6g bearing a methyl group at the 2- or
3-position improved S1P1 agonistic activity by fivefold. Addition-
ally, their selectivity over S1P3 was improved by approximately
Since only 4-phenylbutan-2-ol 12h was commercially available,
the remaining corresponding alcohols 12c–12g and 12i–12n were
HO
a
O
O
b
c
O
O
2
7
3b
HO
O
d
e
CHO
CHO
5b
8
Scheme 2. Synthesis of the key intermediate 8. Reagents: (a) benzyl bromide, potassium carbonate, acetone; (b) MeMgBr/Et2O, THF; (c) EtOAc, HCl-aq; (d) POCl3, DMF; (e)
TFA, PhSMe.

146
H. Kurata et al. / Bioorg. Med. Chem. Lett. 22 (2012) 144–148
Table 2
R3
b
R3
COOH
R2
11c-11g
a
R1
R1
The influence of a methyl group(s) on the alkylene linker between the aromatic rings
on S1P1, S1P3 agonistic activities and in vivo efficacy
OH
OH
CO2Et
R2
12c-12g
13
12i
O
CO2H
R
N
; R1 = p-F, R 2 = R3 = H
; R1 = m-F, R 2 = R3 = H
; R1 = o-F, R 2 = R3 = H
c
d
e
f
CH3
Compound
R
Ca assaya EC50 (nM)
Mouse PLLb ED50 (mg/kg)
po 4 h
; R1 = H, R2 = Me, R3 = H (racemic)
; R1 = H, R2 = H, R3 = Me (racemic)
g
hS1P1
hS1P3
3
6f
0.60
22,000
20,800
>0.3c
0.19
*
*
*
*
R4
4'
4'
c
R4
+
X
HN
O
6g
6h
0.66
O
N
2
O
O
O
O
1
20
20
14,000
N.T.
N.T.
14j ; R4 = Me, X = Cl
15S; (4'-S)
15R; (4'-R)
16j ; R4 = Me,(4'-S)
16k ; R4 = Me,(4'-R)
16l ; R4 = Et, (4'-R)
16m ; R4 = iPr, (4'-R)
14l ; R4 = Et, X = OH
14m ; R4 = iPr, X = Cl
6i
>30,000
a,b
See Table 1.
Decreased by 20% at 0.3 mg/kg.
c
e
X
R4
4'
d
X
R4
OH
O
O
over S1P3 activity than 6c. Additionally, 6k with the S-configura-
tion showed twofold better S1P1 activity, selectivity over S1P3
and in vivo PLL activity than 6j with the R-configuration.
With an aim to further improve the activity and selectivity, we
tested other substituents at the 2-position on the alkylene linker.
Although the selectivity over S1P3 was improved by introducing
a more bulky substituent, the S1P1 activity became weaker as the
substituent became bulkier (6k, 6l and 6m).
Finally, another substituent at the para-position on the terminal
phenyl ring was investigated. 6n bearing a chlorine atom improved
in vivo efficacy compared to 6k by twofold without impairing
selectivity over S1P3 activity. 6n showed efficacy at a dose of
0.095 mg/kg at 24 h after oral administration and 15,000-fold
selectivity for S1P1 over S1P3 agonistic activity in the in vitro assay
(Table 3).
We examined the pharmacokinetic profiles of representative
compounds 6c, 6k and 6n in rats. These three compounds showed
good oral exposure and long half life. The results are summarized
in Table 4 (the pharmacokinetic profile of compound 1 was also re-
ported in a previous paper7b). Compared to compound 1, 6c and 6n
showed longer half-lives (T1/2) and higher plasma concentrations
at 24 h after oral dosing. This suggests that introducing a fluorine
or chlorine atom on the terminal phenyl ring may be beneficial
for the prevention of oxidative metabolism resulting in more po-
tent in vivo PLL activity.
N
2
2
O
17j ; X = F, R 4 = Me, (2-R, 4'-S)
17k ; X = F, R 4 = Me, (2-S, 4'-R)
17l ; X = F, R 4 = Et, (2-S, 4'-R)
12j ; X = F, R 4 = Me, (2-R) (up)
12k ; X = F, R 4 = Me, (2-S) (down)
12l ; X = F, R 4 = Et, (2-S) (down)
17m; X = F, R 4
=
i Pr, (2-R, 4'-R)
12m ; X = F, R 4 i Pr, (2-R) (down)
=
17n ; X = Cl, R4 = Me, (2-S, 4'-R)
12n ; X = Cl, R4 = Me, (2-S) (down)
Scheme 4. Synthesis of 12c–12g and 12i–12n. Reagents: (a) BH3ꢀTHF complex,
THF; (b) LiAlH4, THF; (c) n-BuLi, THF (for 14j and 14m) or DCC, DMAP, CH2Cl2 (for
14l); (d) p-fluorobenzyl bromide (for 17j–17m) or p-chlorobenzyl bromide (for
17n), NaHMDS, THF; (e) LiBH4, THF.
20-fold (6a vs 6f or 6g). In terms of the in vivo PLL activity, 6g
showed an improved in vivo PLL efficacy by twofold (6a vs 6g) at
4 h after oral administration. 6i with two methyl groups at the 2-
position showed less S1P1 agonistic activity than 6g (Table 2).
Substitution of a fluorine atom at the para-position on the ter-
minal phenyl ring was effective for improving in vitro S1P1 agonist
activity as well as in vivo efficacy. Introduction of a methyl group
at the 2-position on the alkylene linker was effective for improving
selectivity over S1P3. Therefore, based on these data, introduction
of both a fluorine atom at the para-position on the terminal phenyl
ring and a methyl group at the 2-position on the alkylene linker
was investigated. As expected, 6j and 6k showed better selectivity
Table 1
The influence of the substitution of a fluorine atom on the terminal phenyl ring on S1P1, S1P3 agonistic activities and in vivo efficacy
O
CO2H
R
N
CH3
Compound
R
Ca assaya EC50 (nM)
Mouse PLLb ED50 (mg/kg)
po 24 h
hS1P1
3.0
hS1P3
5000
3200
po 4 h
0.40
6a
6c
6d
6e
N.T.
0.25
0.69
0.75
*
*
F
F
0.84
0.055
*
*
4.0
2.7
3900
2900
0.15
0.13
F
a
Agonistic activity was evaluated by measuring intracellular Ca2+ concentration stimulation in Chinese Hamster Ovary (CHO) cells stably expressing human S1P1 or S1P3
receptors respectively.
b
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 or 24 h after oral administration of the test compound (N.T. = not tested).

H. Kurata et al. / Bioorg. Med. Chem. Lett. 22 (2012) 144–148
147
Table 3
The influence of the absolute configuration of a methyl group and steric hindrance at the 2-position on the alkylene linker and substituents at the para-position on the terminal
phenyl ring on S1P1, S1P3 agonistic activities and in vivo efficacy
O
CO2H
R
N
CH3
Compound
R
Ca assaya EC50 (nM)
Mouse PLLb ED50 (mg/kg)
po 24 h
hS1P1
hS1P3
F
6j
0.85d
0.37d
1.1
11,000d
0.55
0.22
0.77
0.68
*
*
*
*
R
S
S
R
F
6k
6l
9450d
F
29,300
>30,000
F
6m
4.5
Cl
6n
0.55
8200
0.095
*
S
a,b
See Table 1.
Average of two assays.
d
Table 4
Pharmacokinetic profiles of 1, 6c, 6k and 6n in rata (1 mg/kg)
Compound
AUCinf
(
lg h/mL)
Plasma concentration (ng/mL)
CLtot (mL/min/kg)
T1/2 (h)
Vss (L/kg)
BA (%)
b
Cmax
24 h
1
iv
po
4.4 ( 1.2)
3.8 ( 0.30)
—
—
4.0 ( 1.0)
—
11 ( 4)
17 ( 9)
2.9 ( 0.46)
—
85
181 ( 33)
53 ( 11)
6c
6k
6n
iv
po
5.4 ( 0.81)
5.7 ( 1.7)
2.6c ( 0.33)
2.0c ( 0.22)
—
—
3.2 ( 0.5)
—
21 ( 2)
24 ( 9)
5.3 ( 0.49)
—
106
75c
62
159 ( 79)
88 ( 34)
iv
po
—
—
4.0 ( 0.6)
—
19 ( 2)
57 ( 51)
5.7 ( 0.51)
—
103 ( 5)
69 ( 19)
iv
po
9.0 ( 2.5)
5.5 ( 1.1)
—
—
2.0 ( 0.6)
—
27 ( 6)
20 ( 1)
3.9 ( 0.48)
—
184 ( 46)
87 ( 33)
a
b
c
Values are means of three experiments, standard deviation is given in parentheses.
Tmax for these four compounds are between 4.7 and 6.0 h.
The data until 24 h was adopted.
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In summary, we explored detailed structure–activity relation-
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nistic activity with excellent selectivity over hS1P3 (15,000-fold),
potent in vivo PLL activity in mice (ED50 = 0.095 mg/kg at po 24 h)
and a good pharmacokinetic profile. This study also unveiled that
the introduction of a substituent at the 2-position on the propylene
linker is highly beneficial for the improvement of selectivity over
S1P3. This information should be useful for the consideration of
the binding mode of these compounds with the S1P1 or S1P3
receptor.
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Acknowledgment
The authors thank Mr. T. Shono for his contribution in measur-
ing the PK profile.
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