Discovery of a potent, S1P3-sparing benzothiazole agonist of sphingosine-1-phosphate receptor 1 (S1P1)

Article abstract of DOI:10.1021/ml100228m

Optimization of a benzofuranyl S1P₁ agonist lead compound (3) led to the discovery of 1-(3-fluoro-4-(5-(2-fluorobenzyl)benzo[d]thiazol-2-yl) benzyl)azetidine-3-carboxylic acid (14), a potent S1P₁ agonist with minimal activity at S1P<

Full text of DOI:10.1021/ml100228m

pubs.acs.org/acsmedchemlett
Discovery of a Potent, S1P3-Sparing Benzothiazole Agonist
of Sphingosine-1-Phosphate Receptor 1 (S1P1)
Brian A. Lanman,*,† Victor J. Cee,† Srinivasa R. Cheruku,‡ Mike Frohn,† Jennifer Golden,†
Jian Lin,‡ Mercedes Lobera,‡ Yael Marantz,‡ Kristine M. Muller,† Susana C. Neira,†
Alexander J. Pickrell,† Dalia Rivenzon-Segal,‡ Nili Schutz,‡ Anurag Sharadendu,‡
Xiang Yu,‡ Zhaoda Zhang,‡ Janet Buys,† Mike Fiorino,† Anu Gore,† Michelle Horner,†
Andrea Itano,† Michele McElvain,† Scot Middleton,† Michael Schrag,† Hugo M. Vargas,†
†
†
†
†
†
€
Han Xu, Yang Xu, Xuxia Zhang, Jerry Siu, and Roland W. Burli
†Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320, United States, and
‡EPIX Pharmaceuticals Inc., 167 Worcester Street, Suite 201, Wellesley Hills, Massachusetts 02481, United States
ABSTRACT Optimization of a benzofuranyl S1P1 agonist lead compound (3) led
to the discovery of 1-(3-fluoro-4-(5-(2-fluorobenzyl)benzo[d]thiazol-2-yl)benzyl)-
azetidine-3-carboxylic acid (14), a potent S1P1 agonist with minimal activity at
S1P3. Dosed orally at 0.3 mg/kg, 14 significantly reduced blood lymphocyte counts
24 h postdose and attenuated a delayed type hypersensitivity (DTH) response to
antigen challenge.
KEYWORDS Sphingosine-1-phosphate receptor, S1P1, agonist, inflammation,
lymphocyte
esearch efforts over the past decade have revealed
the lysophospholipid sphingosine-1-phosphate (1, S1P;
Figure 1) to be a pleiotropic modulator of diverse
Human clinical trials have additionally shown dosing of 2
to be associated with transient, dose-dependent decreases in
mean heart rate and asymptomatic atrioventricular blockade.23
Studies of 2 in rodents have revealed lymphocyte sequestration
and heart rate reduction to be driven by distinct S1P receptor
isoforms (S1P1 and S1P3, respectively), however.24-27 These
observations, coupled with the impressive efficacy of 2emerging
from human clinical investigations, have prompted the wide-
spread search for S1P1 agonists with minimal activity at the S1P3
receptor.28,29 In this communication, we describe the dis-
covery of a potent S1P3-sparing S1P1 agonist, benzothiazole
14, as well as its in vivo pharmacological and pharmacokinetic
characteristics.
R
cellular processes including migration, adhesion, proliferation,
and differentiation.1,2 These effects are mediated, at least in
part, by the interaction of S1P with a set of paralogous G protein-
coupled receptors (S1P1-5), which are widely expressed in the
immune, cardiovascular, and central nervous systems.3-5 The
S1P1 receptor, highly expressed in lymphocytes, has recently
been the focus of extensive investigation due to its role in the
regulation of lymphocyte egress from lymph nodes.6-9
Much of this effort has centered on the study of fingolimod
(2, FTY-720),10 a synthetic amino diol prodrug, which is
stereoselectively phosphorylated in vivo by sphingosine kinase
2 to generate the corresponding (S)-phosphate (FTY-720P),
which is a potent agonist of S1P1,3-5. FTY-720P binding to
lymphocyte S1P1 receptors leads to receptor internalization
(RI) and proteolysis.11 Knockout studies in mice have revealed
S1P1 receptor expression in T12,13 and B cells14 to be required
for these lymphocytes to exit secondary lymphoid organs and
enter the circulation; FTY-720P-mediated RI thus leads to
lymphocyte sequestration in secondary lymphoid organs.15
Although FTY-720-mediated lymphocyte sequestration does
not impair antiviral and antibacterial immune responses to
secondary infections in rodent models,16,17 decreased lympho-
cyte recruitment to sites of inflammation has been demon-
strated to moderate immune responses in rodent models of
multiple sclerosis18,19 and organ transplantation.20 Phase III
human clinical trials have recently revealed 2 to be efficacious
in reducing relapse rates in patients with relapsing-remitting
multiple sclerosis.21,22
In prior work, in silico docking of a library of commercially
available molecules with druglike properties into a S1P1
receptor model generated using PREDICT methodology,30
followed by modeling-driven structure-activity relationship
(SAR) studies, led to the identification of a benzofuran-
containing hit series, of which 3 was chosen as the lead
molecule.31 Benzofuran 3 demonstrated double-digit nano-
molar activity at hS1P1 in an assay measuring RI of an hS1P1-
GFP fusion protein in U2OS cells and limited activity at hS1P3
as determined by Ca2þ mobilization in hS1P3- and Gq/i5
-
transfected CHO-K1 cells (Table 1). Oral dosing (1 mg/kg) of
3 in rats resulted in a statistically significant reduction in
circulating lymphocytes 24 h postdose and significantly
Received Date: September 27, 2010
Accepted Date: November 3, 2010
Published on Web Date: November 09, 2010
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Table 2. SAR of 6-Benzyl-Substituted Bicyclic Coresa
hS1P1
RI EC50 (μM)
(% efficacy)
hS1P3
Ca2þ EC50 (μM)
(% efficacy)
compd
W
X
Y
Z
9
10
11
12
13
O
O
N
N
N
C
C
C
C
N
CH
N
C
C
C
N
C
0.013 (110)
0.094 (108)
0.042 (102)
0.741 (110)
1.81 (100)
0.679 (42)
0.542 (87)
1.21 (24)
6.09 (50)
2.71 (48)
Figure 1. S1P, FTY-720, and benzofuran lead 3.
S
Table 1. SAR of 5-Benzyl-Substituted Bicyclic Coresa
CH
CH
a Data represent an average of at least two determinations. Percent
efficacy is reported as in Table 1.
Table 3. Optimization of Benzothiazole Agonistsa
hS1P1 RI
hS1P3 Ca2þ
EC50 (μM)
(% efficacy)
EC50 (μM)
compd
W
X
Y
Z
(% efficacy)
3
4
5
6
7
8
O
O
N
N
N
N
C
C
C
C
C
N
CH
N
C
C
C
C
N
C
0.057 (96)
0.325 (105)
0.221 (84)
>6.25
1.73 (42)
0.354 (77)
3.47 (50)
>25
hS1P1 RI EC50
hS1P3 Ca2þ EC50
S
compd
R
(μM) (% efficacy)
(μM) (% efficacy)
NH
CH
CH
11
14
15
16
17
18
19
20
21
22
23
24
25
H
0.042 (102)
0.042 (115)
0.021 (138)
0.033 (129)
0.042 (186)
0.126 (126)
0.030 (151)
0.026 (118)
0.054 (92)
0.077 (132)
0.042 (121)
0.049 (141)
0.159 (117)
1.21 (24)
3.47 (14)
0.687 (22)
1.91 (37)
1.20 (24)
>25
6.72 (115)
2.89 (53)
>25
2-F
1.11 (43)
3-F
a Data represent an average of at least two determinations. Percent
efficacy is reported relative to 1.00 μM 9 and 0.200 μM S1P for hS1P1 RI
and hS1P3 Ca2þ assays, respectively. (S1P demonstrates 91% efficacy
relative to 1.00 μM 9 in hS1P1 RI studies.) >[Highest concentration
tested] is reported for compounds that do not achieve >10% of control
activity (1.00 μM 9 or 0.200 μM S1P).
4-F
2,4-di-F
2,6-di-F
3,4-di-F
2-Me
3-Me
4-Me
2-Cl
0.967 (19)
2.44 (40)
>2.5
attenuated a delayed type hypersensitivity (DTH) response
to antigen challenge.32 One potential liability of 3 identified
during preclinical toxicology studies was the observation of
proconvulsive activity in rat at oral doses of 40 mg/kg and
higher. Herein, we report on a successful effort to identify
derivatives of 3 lacking this liability.
As proconvulsive activity had not been reported with prior
nonisoform-selective S1P receptor agonists, such activity
was tentatively assumed to be peculiar to benzofuran 3.
An effort was therefore undertaken to identify closely struc-
turally related analogues of 3 that possessed similar or
improved pharmacological profiles but that were devoid of
proconvulsive activity. To this end, a series of compounds
was prepared wherein the benzofuran core of 3 was replaced
with a range of bioisosteric heterocycles (Tables 1 and 2).
Analogues wherein the benzylic substituent of the central
5,6-ring system was located para to the more electronega-
tive substituent (“W”) of the six-membered ring were uni-
formly found to be less potent (4-118-fold) than 3 in the
S1P1 RI assay (Table 1). In contrast, several analogues
wherein the benzylic substituent was located meta to the
1.80 (17)
4.88 (41)
3.88 (35)
>25
3-Cl
4-Cl
a Data represent an average of at least two determinations. Percent
efficacy is reported as in Table 1. >[Highest concentration tested] is
reported for compounds that do not achieve >10% of control activity
(1.00 μM 9 or 0.200 μM S1P).
more electronegative substituent of the six-membered ring
possessed comparable or superior S1P1 activity to 3 (Tabl e 2 ).
Benzothiazole 11 was particularly noteworthy, as it possessed
not only comparable in vitro activity to lead 3 but proved
similarly active in vivo, bringing about statistically significant
lymphocyte depletion upon oral dosing at 1.0 mg/kg.
In an effort to further understand the S1P1 and S1P3 SARs
of benzothiazole 11, a series of analogues was prepared
wherein the terminal phenyl group of 11 was modified with a
range of small lipophilic substituents (Table 3). S1P1 activity
was generally maintained across these analogues; however,
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Figure 2. Compound 14 dosed orally reduces blood lymphocyte
counts in female Lewis rats 24 h postdose (N = 5/group; bars
represent average blood lymphocyte counts þ SEs; circles repre-
sent average plasma concentration ( SEs; *P < 0.05 vs vehicle
by ANOVA/Dunnett's multiple comparison test; vehicle = 20 %
captisol, pH 2).
Figure 3. Compound 14 dosed orally (qd for 10 days) reduces
ovalbumin (OVA)-induced ear swelling in OVA-immunized female
Lewis rats 48 h post-OVA challenge [N = 8/group (vehicle, 0.1, 0.3,
and 1.0 mg/kg), N = 4/group (3.0 mg/kg); bars represent average
change in ear thickness þ SEs; circles represent average blood
lymphocyte counts ( SEs; *P < 0.05 vs vehicle by ANOVA/
Dunnett's multiple comparison test; vehicle = 20% captisol,
pH 2].
4-substitution of the phenyl group with larger substituents led to
moderate decreases in S1P1 activity (22, EC50 = 0.077 μM;
25, EC50 = 0.159 μM), as did 2,6-difluorination (18, EC50
=
Table 4. Pharmacokinetic Parameters for 14
0.126 μM). Substitution of the terminal phenyl group demon-
strated a more varied effect on S1P3 activity: Whereas methyla-
tion had little impact on S1P3 activity (EC50 = 1.80-2.44 μM),
chlorination led to moderate to significant decreases in S1P3
activity (EC50 = 3.88 to >25 μM), and fluorination resulted
species
Cl (L/h/kg)
V
ss (L/kg)
T
1/2 (h)
MRT (h)
% F
rata
NHPb
0.33
0.50
3.3
1.6
7.5
10
68
23
35.2
3.3
a Female Sprague-Dawley (iv: 2 mg/kg, DMSO, N = 3; po: 15 mg/kg,
20% captisol, pH 2, N = 3). b Male Cynomolgus (iv: 4 mg/kg, 20%
captisol, pH 4, N = 3; po: 10 mg/kg, 20% captisol, 1% pluronic F68, 1%
HPMC, pH 2, N = 3).
both in compounds with increased S1P3 activity (15, EC50
=
0.687 μM) and in a compound with no detectable activity at
S1P3 (18, EC50 > 25 μM).
One trend to emerge from these studies was that 2-sub-
stitution of the terminal phenyl group of 11 uniformly led to
moderate, but significant, reductions in S1P3 activity. As a
small lipophilic substituent proved nearly equally effective
to a larger substituent in reducing S1P3 activity (cf. 14, CLog
P = 4.1, and 23, CLog P = 4.6),33 we selected the least
circulating lymphocyte counts, consistent with the cell-
mediated etiology of the DTH immune response.
Pharmacokinetic profiling of 14 in rats and nonhuman
primates (Table 4) revealed 14 to possess acceptable charac-
teristics for further development: 14 demonstrated low clear-
ance (10-19% of liver blood flow), moderate steady state
volumes of distribution (Vss = 1.6-3.3 L/kg), moderate-to-long
mean residence times (3.3-10 h), and acceptable oral bio-
availability (23-68%). In vitro studies established that 14
neither inhibited nor induced human cytochrome P450 en-
zymes, was nonmutagenic (Ames and micronucleus negative),
and did not significantly inhibit the hERG channel in an
electrophysiology assay (PatchXpress;36 IC50 > 10 μM).
Cardiovascular safety studies of 14 in telemetered rats
established a no-effect level for heart rate and mean arterial
pressure changes of 30 mg/kg (po), indicating a wide margin
for S1P3-associated cardiovascular toxicity. Most significantly,
14 did not exhibit proconvulsive activity in Sprague-Dawley
rats (N=3) at a dose of 100 mg/kg when administered orally
once daily for 4 days. The exposure of 14 at the end of the
fourth day (Cmax = 49 ( 10 μg/mL, AUC0-24 = 987 ( 185
lipophilic analogue, 2-fluorobenzyl benzothiazole 14 (RI EC50
=
0.042 μM, Ca2þ EC50 = 3.47 μM), for further in vitro and in vivo
profiling.34
Dosed orally at 0.3, 1.0, and 3.0 mg/kg in Lewis rats, 14
produced a dose-dependent reduction in circulating blood
lymphocytes 24 h postdose, consistent with S1P1 agonism
(Figure 2). Statistical significance (P < 0.05) was achieved at
a dose of 0.3 mg/kg (34% reduction in lymphocytes vs
vehicle), and a 3.0 mg/kg dose resulted in near maximal
lymphopenia (74% reduction in lymphocytes vs vehicle).35
Compound 14 was subsequently investigated in a DTH
antigen challenge model, an in vivo model of cell-mediated
immune responses. In this study, Lewis rats were orally dosed
with 0.1, 0.3, 1.0, or 3.0 mg/kg 14 or vehicle once daily over
the course of 10 days. One day after initiating dosing, rats
were immunized with a mixture of ovalbumin and complete
Freund's adjuvant. One week later, the immunized rats were
rechallenged with ovalbumin by intracutaneous injection into
one ear. After an additional 2 days, differential swelling of the
inoculated ear (determined by change in ear thickness) and
circulating blood lymphocyte counts were measured. As seen in
Figure 3, a statistically significant (P < 0.05) reduction in ear
swelling was observed at doses of 0.3 mg/kg and higher.
Furthermore, reduced ear swelling was found to closely track
μg h/mL) was significantly higher (3-fold Cmax, 40-fold
3
AUC0-24) than the exposure at which proconvulsive activity
was observed with benzofuran 3. As the plasma free fractions37 of
14 (2.1%) and 3 (1.5%) in rat were similar, the lack of proconvul-
sive activity of 14 relative to 3 is currently not well understood.
The synthesis of benzothiazole 14 is described in Scheme 1.
Aryl bromide 26, prepared in one step from commercially
available 3-fluoro-4-bromobenzaldehyde, was treated with
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Scheme 1. Synthesis of 14a
Vounatsos for large-scale synthesis (14 and methyl azetidine-3-carboxy-
late hydrochloride); and Oren Becker, Pini Orbach, Ashis Saha, and
Sharon Shacham for their leadership of research efforts at EPIX Phar-
maceuticals, Inc. Dilara McCauley and Ashis Saha are acknowledged
for their leadership of prior studies leading to the identification of
benzofuran 3.
REFERENCES
(1)
Hannun, Y. A.; Obeid, L. M. Principles of Bioactive Lipid
Signaling: Lessons from Sphingolipids. Nature Rev. Mol. Cell.
Biol. 2008, 9, 139–150.
a Reagents and conditions: (a) n-BuLi, 27, THF, -78 °C, 63%.
(b) Na2CO3, DMF, 110 °C, 93%. (c) 5 N HCl, THF, 55 °C, 100%. (d) Methyl
azetidine-3-carboxylate hydrochloride, AcOH, DIPEA, NaBH3CN, MeOH/
CH2Cl2, 71%. (e) Bis(di-tert-butyl(phenyl)phosphine) palladium dichlo-
ride (5 mol %), (2-fluorobenzyl)zinc chloride, THF, 85%. (f) LiOH, THF/
H2O, then HCl and pH 6 sodium phosphate buffer, 99%.
(2)
Kihara, A.; Mitsutake, S.; Mizutani, Y.; Igarashi, Y. Metabolism
and Biological Functions of Two Phosphorylated Sphingo-
lipids, Sphingosine-1-Phosphate and Ceramide 1-Phosphate.
Prog. Lipid Res. 2007, 46, 126–144.
Rosen, H.; Gonzalez-Cabrera, P. J.; Sanna, M. G.; Brown, S.
Sphingosine 1-Phosphate Receptor Signaling. Annu. Rev.
Biochem. 2009, 78, 743–768.
Cyster, J. G. Chemokines, Spingosine-1-Phosphate, and Cell
Migration in Secondary Lymphoid Organs. Annu. Rev. Immunol.
2005, 23, 127–159.
Rosen, H.; Goetzl, E. J. Sphingosine 1-Phosphate and its Recep-
tors: An Autocrine and Paracrine Network. Nature Rev. Immunol.
2005, 5, 560–570.
Marsolais, D.; Rosen, H. Chemical Modulators of Sphingo-
sine-1-Phosphate Receptors as Barrier-Oriented Therapeutic
Molecules. Nature Rev. Drug Discovery 2009, 8, 297–307.
Brinkmann, V. Sphingosine 1-Phosphate Receptors in Health and
Disease: Mechanistic Insights from Gene Deletion Studies and
Reverse Pharmacology. Pharmacol. Ther. 2007, 115, 84–105.
Gardell, S. E.; Dubin, A. E.; Chun, J. Emerging Medicinal Roles
for Lysophospholipid Signaling. Trends Mol. Med. 2006, 12,
65–75.
Mandala, S.; Hajdu, R.; Bergstrom, J.; Quackenbush, E.; Xie,
J.; Milligan, J.; Thornton, R.; Shei, G.-J.; Card, D.; Keohane, C.;
Rosenbach, M.; Hale, J.; Lynch, C. L.; Rupprecht, K.; Parsons,
W.; Rosen, H. Alteration of Lymphocyte Trafficking by Sphin-
gosine-1-Phosphate Receptor Agonists. Science 2002, 296,
346–349.
(3)
(4)
(5)
(6)
(7)
(8)
(9)
n-butyllithium at -78 °C, and 2-fluoro-5-bromophenyl isothio-
cyanate 27 was then added to the resulting aryl lithium species,
generating the corresponding thioamide. Incubation of the
thioamide with a suspension of sodium carbonate in hot DMF
resulted in clean cyclization to benzothiazole 28. Exposure of 28
to HCl in warm THF resulted in cleavage of the acetal protecting
group; reductive amination of the resulting aldehyde subse-
quently provided methyl azetidine carboxylate 29. Negishi
coupling of 29 with (2-fluorobenzyl)zinc chloride delivered
5-benzylbenzothiazole 30, and saponification of 30 followed
by pH adjustment of the resulting reaction mixture resulted in
the precipitation of benzothiazole 14 as its zwitterion.
In summary, replacement of the benzofuranyl core of a
novel S1P1 agonist (3) with a series of bioisosteric heterocycles
led to the identification of benzothiazole 11, which retained
both the S1P1 activity and the S1P3 selectivityof 3. Subsequent
substitution of the terminal phenyl group of 11 with a range of
small lipophilic substituents led to the discovery of 2-fluoro-
benzyl benzothiazole 14, which was found to be a potent
S1P1 agonist (EC50 = 0.042 μM) with reduced activity at S1P3
(EC50 = 3.47 μM). Oral dosing of 14 in rats (0.3 mg/kg)
resulted in a statistically significant reduction in circulating
lymphocytes 24 h postdose, as well as a statistically significant
attenuation of a DTH response to antigen challenge. Further-
more, benzothiazole 14 was devoid of the proconvulsive activity
of 3 at doses of up to 100 mg/kg. In vitro and in vivo pharma-
cokinetic studies and safety studies have established that 14
possesses acceptable properties for further development.
(10) Adachi, K.; Chiba, K. FTY720 Story. Its Discovery and the
Following Accelerated Development of Sphingosine 1-Phos-
phate Receptor Agonists as Immunomodulators Based on
Reverse Pharmacology. Perspect. Med. Chem. 2007, 1, 11–23.
(11) Oo, M. L.; Thangada, S.; Wu, M.-T.; Liu, C. H.; Macdonald, T. L.;
Lynch, K. R.; Lin, C.-Y.; Hla, T. Immunosuppressive and Anti-
angiogenic Sphingosine 1-Phosphate Receptor-1 Agonists
Induce Ubiquitinylation and Proteasomal Degradation of
the Receptor. J. Biol. Chem. 2007, 282, 9082–9089.
(12) Matloubian, M.; Lo, C. G.; Cinamon, G.; Lesneski, M. J.; Xu, Y.;
Brinkmann, V.; Allende, M. L.; Proia, R. L.; Cyster, J. G. Lym-
phocyte Egress from Thymus and Peripheral Lymphoid Organs
is Dependent on S1P Receptor 1. Nature 2004, 427, 355–360.
(13) Allende, M. L.; Dreier, J. L.; Mandala, S.; Proia, R. L. Expres-
sion of the Sphingosine 1-Phosphate Receptor, S1P1, on
T-cells Controls Thymic Emigration. J. Biol. Chem. 2004,
279, 15396–15401.
(14) Kabashima, K.; Haynes, N. M.; Xu, Y.; Nutt, S. L.; Allende,
M. L.; Proia, R. L.; Cyster, J. G. Plasma Cell S1P1 Expression
Determines Secondary Lymphoid Organ Retention Versus
Bone Marrow Tropism. J. Exp. Med. 2006, 203, 2683–2690.
(15) Chiba, K.; Yanagawa, Y.; Masubuchi, Y.; Kataoka, H.; Kawaguchi,
T.; Ohtsuki, M.; Hoshino, Y. FTY720, a Novel Immunosuppres-
sant, Induces Sequestration of Circulating Mature Lymphocytes
SUPPORTING INFORMATION AVAILABLE Statistical anal-
ysis of data presented in Tables 1-3, experimental procedures and
characterization data for 3-25, and details of in vitro and in vivo
assays. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author: *Tel: 805-313-5777. Fax: 805-480-
1337. E-mail: blanman@amgen.com.
ACKNOWLEDGMENT We thank Ronya Shatila and Beth Tominey
for in vivo PKDM studies; Brenda Burke, Bobby Riahi, and Filisaty
r
2010 American Chemical Society
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pubs.acs.org/acsmedchemlett
by Acceleration of Lymphocyte Homing in Rats. I. FTY720
Selectively Decreases the Number of Circulating Mature Lym-
phocytes by Acceleration of Lymphocyte Homing. J. Immunol.
1998, 160, 5037–5044.
(27) Brossard, P.;Hofmann, S.;Cavallaro, M.;Halabi, A.;Dingemanse,
J. Entry-into-Humans Study with ACT-128800, A Selective S1P1
Receptor Agonist: Tolerability, Safety, Pharmacokinetics, and
Pharmacodynamics. Clin. Pharmacol. Ther. 2009, 85, S63–S64.
(28) Buzard, D. J.; Thatte, J.; Lerner, M.; Edwards, J.; Jones, R. M.
Recent Progress in the Development of Selective S1P1
Receptor Agonists for the Treatment of Inflammatory and
Autoimmune Disorders. Expert Opin. Ther. Patents 2008, 18,
11 41 –1159.
(16) Pinschewer, D. D.; Ochsenbein, A. F.; Odermatt, B.; Brink-
mann, V.; Hengartner, H.; Zinkernagel, R. M. FTY720 Im-
munosuppression Impairs Effector T Cell Peripheral Homing
Without Affecting Induction, Expansion, and Memory. J. Immunol.
2000, 164, 5761–5770.
ꢀ
(29) Cooke, N.; Zecri, F. Sphingosine 1-Phosphate Type 1 Receptor
€
(17) Kursar, M.; Janner, N.; Pfeffer, K.; Brinkmann, V.; Kaufmann,
Modulators: Recent Advances and Therapeutic Potential.
Annu. Rep. Med. Chem. 2007, 42, 245–263. For reports from
2008 to the present and modeling-based approaches to ratio-
nalizing isoform selectivity, see the Supporting Information.
(30) Shacham, S.; Marantz, Y.; Bar-Haim, S.; Kalid, O.; Warshaviak,
D.; Avisar, N.; Inbal, B.; Heifetz, A.; Fichman, M.; Topf, M.; Naor,
Z.; Noiman, S.; Becker, O. M. PREDICT Modeling and In-Silico
Screening for G-Protein Coupled Receptors. Proteins 2004, 57,
51–86.
€
S. H. E.; Mittrucker, H.-W. Requirement of Secondary Lym-
phoid Tissues for the Induction of Primary and Secondary T
cell Responses Against Listeria monocytogenes. Eur. J. Immu-
nol. 2008, 38, 127–138.
(18) Kataoka, H.; Sugahara, K.; Shimano, K.; Teshima, K.; Koyama,
M.; Fukunari, A.; Chiba, K. FTY720, Sphingosine 1-Phosphate
Receptor Modulator, Ameliorates Experimental Autoimmune
Encephalomyelitis by Inhibition of T Cell Infiltration. Cell. Mol.
Immunol. 2005, 2, 439–448.
(31) Saha, A. K.; Yu, X.; Lin, J.; Lobera, M.; Sharadendu, A.; Cheruku,
S.; Schutz, N.; Segal, D.; Marantz, Y.; McCauley, D.; Buys, J.;
Horner, M.; Salyers, K.; Schrag, M.; Vargas, H. M.; Xu, Y.;
McElvain, M.; Xu, H. Benzofuran-based Sphingosine-1 Phos-
phate Receptor-1 (S1P1) Agonists: Discovery of a Potent,
Selective and Orally Bioavailable Preclinical Lead Molecule.
ACS Med. Chem. Lett., in press.
(32) Ovalbumin/complete Freund's adjuvant. For experimental
protocol, see the Supporting Information.
(33) ChemBioDraw Ultra, version 11.0, Cambridgesoft, 100
CambridgePark Drive, Cambridge, MA 02140.
(19) Fujino, M.; Funeshima, N.; Kitazawa, Y.; Kimura, H.; Ame-
miya, H.; Suzuki, S.; Li, X.-K. Amelioration of Experimental
Autoimmune Encephalomyelitis in Lewis Rats by FTY720
Treatment. J. Pharmacol. Exp. Ther. 2003, 305, 70–77.
(20) Chiba, K.; Hoshino, Y.; Suzuki, C.; Masubuchi, Y.; Yanagawa, Y.;
Ohtsuki, M.; Sasaki, S.; Fujita, T. FTY720, A Novel Immunosup-
pressant Possessing Unique Mechanisms. I. Prolongation of Skin
Allograft Survival and Synergistic Effect in Combination with
Cyclosporine in Rats. Transplant. Proc. 1996, 28, 1056–1059.
(21) Kappos, L.; Radue, E.-W.; O'Connor, P.; Polman, C.; Hohlfeld,
R.; Calabresi, P; Selmaj, K.; Agoropoulou, C.; Leyk, M.; Zhang-
Auberson, L.; Burtin, P. A Placebo-Controlled Trial of Oral
Fingolimod in Relapsing Multiple Sclerosis. N. Engl. J. Med.
2010, 362, 387–401.
(34) Dosed orally (1 mg/kg) in female Lewis rats, 14, 20, and 23
were similarly efficacious in reducing blood lymphocyte
counts 24 h postdose (63-73% reduction).
(35) S1P1 agonists have previously been reported to produce a
maximal ∼70% reduction in lymphocyte count: Hale, J. J.;
Lynch, C. L.; Neway, W.; Mills, S. G.; Hajdu, R.; Keohane, C. A.;
Rosenbach, M. J.; Milligan, J. A.; Shei, G.-J.; Parent, S. A.;
Chrebet, G.; Bergstrom, J.; Card, D.; Ferrer, M.; Hodder, P.;
Strulovici, B.; Rosen, H.; Mandala, S. A Rational Utilization of
High-Throughput Screening Affords Selective, Orally Bio-
available 1-Benzyl-3-carboxyazetidine Sphingosine-1-phos-
phate-1 Receptor Agonists. J. Med. Chem. 2004, 47, 6662–
6665.
(22) Cohen, J. A.; Barkhof, F.; Comi, G.; Hartung, H.-P.; Khatri,
B. O.; Montalban, X.; Pelletier, J.; Capra, R.; Gallo, P.; Izquierdo,
G.; Tiel-Wilck, K.; de Vera, A.; Jin, J.; Stites, T.; Wu, S.; Aradhye, S.;
Kappos, L. Oral Fingolimod or Intramuscular Interferon for
Relapsing Multiple Sclerosis. N. Engl. J. Med. 2010, 362, 402–415.
(23) See refs 21, 22, and Schmouder, R.; Serra, D.; Wang, Y.;
Kovarik, J. M.; DiMarco, J.; Hunt, T. L.; Bastien, M.-C. FTY720:
Placebo-Controlled Study of the Effect on Cardiac Rate and
Rhythm in Healthy Subjects. J. Clin. Pharmacol. 2006, 46,
895–904.
(24) Forrest, M.; Sun, S.-Y.; Hajdu, R.; Bergstrom, J.; Card, D.;
Doherty, G.; Hale, J.; Keohane, C.; Meyers, C.; Milligan, J.;
Mills, S.; Nomura, N.; Rosen, H.; Rosenbach, M.; Shei, G.-J.;
Singer, I. I.; Tian, M.; West, S.; White, V.; Xie, J.; Proia, R. L.;
Mandala, S. Immune Cell Regulation and Cardiovascular
Effects of Sphingosine-1-Phosphate Receptor Agonists in Rodents
are Mediated via Distinct Receptor Subtypes. J. Pharmacol. Exp.
Ther. 2004, 309, 758–768.
(36) PatchXpress 7000A Automated Parallel Patch-Clamp System,
Molecular Devices, 1311 Orleans Drive, Sunnyvale, CA 94089.
(37) Determined by ultracentrifugation at 5.0 μg/mL test com-
pound concentration.
(25) Sanna, M. G.; Liao, J.; Jo, E.; Alfonso, C.; Ahn, M.-Y.; Peterson,
M. S.; Webb, B.; Lefebvre, S.; Chun, J.; Gray, N.; Rosen, H.
Sphingosine 1-Phosphate (S1P) Receptor Subtypes S1P1 and
S1P3, Respectively, Regulate Lymphocyte Recirculation and
Heart Rate. J. Biol. Chem. 2004, 279, 13839–13848.
(26) Recent research has suggested that S1P1 rather than S1P3
€
mediates heart rate reduction in humans: Gergely, P.; Wallstrom,
ꢀ
E.; Nuesslein-Hildesheim, B.; Bruns, C.; Zecri, F.; Cooke, N.;
Traebert, M.; Tuntland, T.; Rosenberg, M.; Saltzman, M. Phase I
study with the selective S1P1/S1P5 receptor modulator BAF312
indicates that S1P1 rather than S1P3 mediates transient heart rate
reduction in humans. Mult. Scler. 2009, 15, S125–S126. See also
ref 27.
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2010 American Chemical Society
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DOI: 10.1021/ml100228m ACS Med. Chem. Lett. 2011, 2, 102–106
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