Structure-activity relationship studies of sphingosine-1-phosphate receptor agonists with N-cinnamyl-β-alanine moiety

Article abstract of DOI:10.1016/j.bmcl.2011.01.029

Structure-activity relationship of sphingosine-1-phosphate receptor agonist was examined. In terms of reducing the flexibility of molecule, hit compound 1 was modified to improve S1P₁ agonistic activity as well as selectivity over S1P₃ agonistic activity. Novel S1P agonists with cinnamyl scaffold or 1,2,5,6-tetrahydropyridine scaffold were identified.

Full text of DOI:10.1016/j.bmcl.2011.01.029

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1390–1393
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationship studies of sphingosine-1-phosphate
receptor agonists with N-cinnamyl-b-alanine moiety
Haruto Kurata a, , Kazuhiro Otsuki a, Kensuke Kusumi a, Masakuni Kurono a, Masahiko Terakado a,
⇑
Takuya Seko a, 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 agonist was examined. In terms of
reducing the flexibility of molecule, hit compound 1 was modified to improve S1P1 agonistic activity
as well as selectivity over S1P3 agonistic activity. Novel S1P agonists with cinnamyl scaffold or 1,2,5,6-
tetrahydropyridine scaffold were identified.
Received 25 November 2010
Revised 6 January 2011
Accepted 7 January 2011
Available online 11 January 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
S1P agonist
Immunomodulator
Sphingosine-1-phosphate (S1P) exerts a variety of biological
activities, including vascular maturation and cell survival.1 Five
S1P receptor subtypes have been known as S1P1–5, respectively.
One significant achievement in S1P research field was published
in 2002. Lynch and co-workers reported that FTY720 (Fingolimod,
Fig. 1), which was developed as a novel immunomodulator2 and
was recently approved in Russia and USA for the treatment of mul-
tiple sclerosis, is metabolized across species to a monophosphate
ester, which can activate four S1P receptors (S1P1,3–5) to sequester
lymphocytes from circulation to secondary lymph tissue compart-
ment.3 It is also reported that S1P1 is essential for lymphocyte
recirculation since S1P1 modulates egress from thymus and
peripheral lymphoid organs.4
relationship around 1 to improve S1P1 agonistic activity and selec-
tivity against S1P3.
Compounds in this report were synthesized in Schemes 1–3 as
shown below. Compound
1 was prepared in 21% yield and
compounds 9a–9c were synthesized by using a solid-phase method7
from 4-(3-aminopropyl)phenol 2. Starting from compound 2, N-Boc-
b-alanine derivative 5 was prepared in 56% yield. 2-Chlorotrityl resin
was reacted with 5 and treated with the corresponding alcohol in the
presence of tri-n-butylphosphine and N,N,N0,N0-tetramethylazo-
dicarboxamide (TMAD). The treatment with the acetic acid and
2,2,2-trifluoroethanol yielded Boc-protected b-alanine 8a–8c, which
was treated with HCl/EtOAc to give 9a–9c (Scheme 1).
Cinnamyl derivatives 12a–12d were synthesized by reductive
alkylation of b-alanine with aldehydes8 11a–11d in acceptable
yields. Cinnamyl aldehyde 11a was derived by reduction of the
corresponding ester by LiAlH4 in presence of Et2NH9 in 17% yield.
(4-Hydroxy)-acetophenone 13c was treated with diethyl (cyanom-
It was reported that S1P3 agonism does not relate to lympho-
cyte recirculation but links to bradycardia in rodents.5 Asymptom-
atic bradycardia was also reported in clinical studies of FTY720.6
Herein, we report our result to identify orally available S1P1
agonists without S1P3 agonism.
ethyl)-phosphonate and the resulting
a,b-unsaturated nitrile was
High Throughput Screening (HTS) campaign of our historical
compound library yielded a hit compound 1, which showed a sub-
micromolar human S1P1 agonistic activity with human S1P3 and
human S1P5 agonistic activities. Additionally, oral administration
of 1 in mice could induce peripheral lymphocyte lowering (PLL)
with 50% effective dose (ED50) of 26 mg/kg at 4 h after oral dosing
(Fig. 2). Therefore, we decided to investigate the structure–activity
converted to the corresponding aldehyde, which was treated with
alkyl bromide and each regioiosomer generated under the reaction
condition could be separated by column chromatography. The
H3C
NH2
OR
OH
R = H; FTY720 (Fingolimod)
R = PO3H2
HCl
⇑
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).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.029

H. Kurata et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1390–1393
1391
Ca Assay
EC50 (uM)
Mouse PLL
ED50 (mg/kg)
p.o. 4 hr
26
hS1P1
0.16
hS1P2
>10.0
hS1P3
4.2
hS1P5
0.17
O
H
N
CO2 H
Hit compound 1
Figure 2. Structure and pharmacological profiles of hit compound 1.
HO
b
Boc
N
HO
a
CO2R
+
t
NH2
CO2 Bu
3m
(R = t-Bu)
4
2
d
c
O
H
N
CO2H
HCl
1
HO
f
e
a
b
Boc
N
+
2
CO2R
3n
(R = Me)
CO2H
5
HO
O
g
h
Boc
Boc
n
N
O
N
O
Pol
Pol
O
O
6
7a-c
i
O
O
Boc
N
H
N
HCl
n
n
CO2H
CO2H
8a-c
9a-c
a; n = 1
b; n = 2
c; n = 4
Scheme 1. Reagent: (a) MeOH; (b) Boc2O, THF; (c) Ph(CH2)5Br, K2CO3, DMF; (d) HCl/1,4-dioxane; (e) NaOH-aq, MeOH, THF; (f) Pol-Cl (Pol = 2-chlorotrityl resin), i-Pr2NEt,
CH2Cl2; (g) Ph–(CH2)n–CH2CH2OH, n-Bu3P, TMAD, CH2Cl2, THF; (h) CF3CH2OH, AcOH, CH2Cl2; (i) HCl/EtOAc.
separated trans- and cis-
were reacted with b-alanine to yield 12c and 12d, respectively.
(Scheme 2).
a
,b-unsaturated aldehydes 11c and 11d
activity and oral exposure level was investigated. Although com-
pound 1 showed the highest activity against S1P1, shorter ana-
logues 9a and 9b as well as longer analogue 9c retained the S1P1
agonistic activity. On the other hand, compound 9a showed higher
oral exposure level in rat than compound 1 (Table 1), which sup-
ports that the restriction of conformational flexibility improves
the oral absorption.10 We decided to use propylene linker for the
further SAR studies.
Tetrahydropyridine derivative 18 and piperidine derivative 19
were prepared via tetrahydro pyridine derivative 17, which was
obtained from the corresponding piperidone 15. Piperidone 15
was reacted with lithiated phenylbromide 14 and then converted
to tetrahydro pyridine 17 by HCl deprotection of Boc group fol-
lowed by dehydration and Michael addition to tert-butyl acrylate
in 52% yield. Tetrahydropyridine derivative 17 was converted to
18 by the treatment with trifluoroacetic acid (TFA) in 82% yield,
and 19 by hydrogenation and subsequent treatment with TFA in
52% yield (Scheme 3).
Piperidine derivative 24 was prepared from 3-hydroxypiperi-
dine 20. Piperidine derivative 20 was alkylated and oxidised to give
21 in 77% yield. On the other hand, phosphonium salt 23 was ob-
tained from benzyl alcohol 22 in 94% yield. Phosphonium salt 23
was treated with dimsyl anion to afford a phosphonium ylide
and reacted with ketone 21 to yield the condensed product in
regioisomeric mixture, which was deprotected by HCl and then
separated by column chromatography to give the trans isomer 24
(Scheme 3).
Next we modified the propylene linker between the internal
phenyl ring and nitrogen (NH) in compound 9a by introducing a
double bond. Cinnamylamino compound 12a significantly im-
proved not only S1P1 agonist activity but also selectivity over
S1P3. Hale et al. reported the S1P1 selective agonists with benzyl
amine moiety at the
this information, it is supposed that the
c
-position of carboxylic acid.11 By combining
-electron density at this
p
position might be important for S1P1 agonist activity (Table 2).
Introducing a methyl group on the double bond of the allyl
group of 12a was examined. Whereas compound 12b with methyl
group at the b-position of amine showed comparable S1P1 agonis-
tic activity and selectivity over S1P3, compound 12c with methyl
group at the c-position improved both S1P1 activity and selectivity.
Additionally, the activity to reduce the peripheral blood
lymphocyte count was enhanced significantly. Compound 12d
with cis-double bond showed less S1P1 agonistic activity than
The effect of the length of alkylene linker between terminal
phenyl and internal phenyl ring of hit compound 1 on its in vitro

1392
H. Kurata et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1390–1393
Table 1
c
b
a
HO
The influence of length of linker towards S1P1 and S1P3 agonistic activities and oral
exposure levels in rat
O
CO2H
CHO
10
11a
O
H
N
n
CO2H
d
O
H
N
Ca assaya EC50
M)
Pharmacokinetic propertiesb in rat
(30 mg/kg p.o.)
CO2H
Compound
n
(
l
12a
hS1P1 hS1P3 AUCinf
lg h/mL Cmax
l
g/mL T1/2
h
g
f
e
d
R2
CHO
R1
9a
9b
1
1
2
3
4
2.6
1.5
0.16
2.7
9.0
>10
4.2
174 ( 22)
N.T.
11 ( 1.1)
N.T.
28 ( 6.4)
N.T.
2.1 ( 0.23)
N.T.
3.8 ( 0.4)
N.T.
3.8 ( 0.1)
N.T.
O
HO
R1
O
13b-c
9c
>10
11b-d
a
Agonistic activity was evaluated by the measurement of intracellular calcium
concentration stimulated by the addition of each compound for human S1P1 or
human S1P3 receptors stably expressed in Chinese Hamster Ovary (CHO) cell,
respectively.
Values are means of three experiments, standard deviation is given in paren-
theses (N.T. = not tested).
h
R2
O
b; R1=H, R2=Me, trans
c; R1=Me, R2=H, trans
d; R1=Me, R2=H, cis
H
N
b
CO2H
HCl
R1
12b-d
Scheme 2. Reagent: (a) SOCl2, MeOH; (b) Ph(CH2)3OH, Ph3P, DEAD, THF; (c) LiAlH4,
Et2NH, n-hexane; (d) (i) H2NCH2CH2CO2H, NaOH, HC(OMe)3, MeOH, THF; (ii)
NaBH4; (e) (EtO)2P(O)CHR2CN (b: R2 = Me, c: R2 = H), NaH, THF; (f) DIBAH, THF; (g)
(i) Ph(CH2)3Br, K2CO3, DMF; (ii) separation of regioisomer 11c and 11d; (h) HCl-aq/
THF.
Table 2
Replacement of propylene linker to allyl linker
R =
*
Compound Structure
Ca assaya EC50 Mouse PLLb
M) ED50 (mg/kg)
(l
hS1P1 hS1P3 p.o. 4 h
O
O
R
H
N
9a
2.6
9.0
N.T.
4.6
N
15
Boc
CO2H
CO2H
O
O
R
a
H
N
O
12a
0.070 3.3
HO
Br
N
Boc
a
14
See Table 1.
16
b
Individual data points for dose–titrations were the average percentage decrease
of peripheral blood lymphopocyte counts in n = 5 animals versus control (n = 5) 4 h
O
b
c
d
after oral administration of the test compound (N.T. = not tested).
N
t
CO2 Bu
17
e
O
O
Table 3
17
Addition of a methyl group on double bond
TFA
N
N
R =
CO2H
18
19
*
f
e
Compound Structure
Ca assaya EC50 Mouse PLLb
M) ED50 (mg/kg)
17
TFA
CO2H
(l
hS1P1 hS1P3 p.o. 4 h
O
R
h
H
N
g
12b
0.033
1.9 N.T.
N
CO2H
CO2H
NH
t
O
HO
CO2 Bu
O
R
21
20
H
N
12c
0.021 >10.0 0.69
Br
j
i
H
N
O
O
O
P+
R
CO2H
12d
0.93
>10.0 N.T.
OH
l
22
23
a,b
See Tables 1 and 2.
k
O
+
23
21
N
HCl
COOH
trans-congener 12c (Table 3), which indicates the trans configura-
tion might be beneficial to assist the basic nitrogen for interacting
with S1P1 receptor. This unexpected result prompted us to investi-
gate further active conformations of compound 12a.
Compound 12a should have two distinct conformations around
the allyl amine linker: extended conformer A and folded conformer
24
Scheme 3. Reagent: (a) n-BuLi, THF; (b) HCl/1,4-dioxane, CH2Cl2; (c) Et3SiH, TFA,
CH2Cl2; (d) CH2@CHCO2But, iPr2NEt, DMF; (e) TFA, CH2Cl2; (f) H2, 10% Pd-C, EtOAc;
(g) CH2@CHCO2tBu, MeOH; (h) (COCl)2, DMSO, Et3N, CH2Cl2; (i) CBr4, Ph3P, CH2Cl2;
(j) Ph3P, PhMe; (k) NaH, DMSO; (l) (i) HCl/EtOAc, (ii) separation of regioisomers.

H. Kurata et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1390–1393
1393
and selectivity over S1P3. Compound 12c also showed superior
pharmacokinetic property with long half-time (T1/2 = 7.4 h) in rat
R =
*
(data not shown). During the SAR study, the importance of p-elec-
tron density between the central benzene ring and nitrogen atom
was also determined. Furthermore, we conclude that the active
conformation of compound 12a would be the folded conformer
18. Further optimisation of compounds 12c and 18 will be reported
in due course.
O
R
O
R
H
N
CO2H
HN
CO2H
12a
extended
conformer A
folded
conformer B
Acknowledgements
O
O
R
R
Authors thank Mr. T. Shono and Mr. N. Matsumura for their con-
tribution on measuring the PK profile and acknowledge Dr. I.C.
Choong, Dr. H. Nakai and Dr. T. Kondo for preparation of this
manuscript.
N
CO2H
N
CO2H
24
18
Figure 3. Conformational relationship between compounds 18 and 24.
Supplementary data
Table 4
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.01.029. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
Cyclic derivatives of compound 12a
R =
*
Compound Structure
Ca assaya EC50 Mouse PLLb
(
l
M)
ED50 (mg/kg)
References and notes
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R
18
0.045
1.5
1.2 1.4
>10.0 N.T.
>10.0 N.T.
N
CO H
2
O
R
24
N
CO H
2
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19
1.6
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N
H
CO2
a,b
See Tables 1 and 2.
B (Fig. 3). Compounds 24 and 18 were designed to fix the extended
conformation A and folded conformer B, respectively.
As a result, compound 18 showed much stronger S1P1 activity
than compound 24 (Table 4). This data strongly suggests that the
folded conformer B would be an active conformation of compound
12a. Additionally, the activity of compound 19, having a single
bond instead of double bond in the piperidine ring compared to
compound 18, was weaker than compound 18, which indicates
again that the p-electron density at this position might be impor-
tant to activate S1P1 receptor.
In summary, we explored SAR of hit compound 1 to identify a
cinnamyl derivative 12a with improved in vitro S1P1 agonist
activity as well as selectivity over S1P3. Further modification of
compound 12a led to compound 12c with strong S1P1 agonism