Discovery of potent 3,5-diphenyl-1,2,4-oxadiazole sphingosine-1-phosphate-1 (S1P1) receptor agonists with exceptional selectivity against S1P2 and S1P3

Article abstract of DOI:10.1021/jm0503244

A class of 3,5-diphenyl-1,2,4-oxadiazole based compounds have been identified as potent sphingosine-1-phosphate-1 (S1P₁) receptor agonists with minimal affinity for the S1P₂ and S1P₃ receptor subtypes. Analogue 26 (S1P₁ IC₅₀ = 0.6 nM) has an excellent pharmacokinetics profile in the rat and dog and is efficacious in a rat skin transplant model, indicating that S1P₃ receptor agonism is not a component of immunosuppressive efficacy.

Full text of DOI:10.1021/jm0503244

© Copyright 2005 by the American Chemical Society
Volume 48, Number 20
October 6, 2005
Letters
has inspired considerable interest in agonists and antag-
onists of sphingosine-1-phosphate (S1P) receptors.2 S1P
is a bioactive lysolipid with pleiotropic functions medi-
ated via agonism of a family of G-protein-coupled
receptors, S1P1-5.3 It has been demonstrated that 1 is
Discovery of Potent
3,5-Diphenyl-1,2,4-oxadiazole
Sphingosine-1-phosphate-1 (S1P1)
Receptor Agonists with Exceptional
Selectivity against S1P2 and S1P3
Zhen Li,*,† Weirong Chen,‡ Jeffrey J. Hale,†
Christopher L. Lynch, Sander G. Mills,†
Richard Hajdu,§ Carol Ann Keohane,§
Mark J. Rosenbach,§ James A. Milligan,§
Gan-Ju Shei,§ Gary Chrebet,§ Stephen A. Parent,§
James Bergstrom,§ Deborah Card,§ Michael Forrest,§
Elizabeth J. Quackenbush,§ L. Alexandra Wickham,§
Hugo Vargas,# Rose M. Evans,# Hugh Rosen,| and
Suzanne Mandala§
metabolized across species to a monophosphate ester (2),
which is a high-affinity ligand for S1P1,3-5 but not S1P2.4
Preclinical studies with 1 have revealed that its physi-
ological effects include a redistribution of lymphocytes
from blood to secondary lymphoid organs and regulation
of cardiovascular function, with the former being a
driver of immunosuppressive efficacy.5 S1P1 receptor
agonism has been shown to correlate with lymphocyte
recirculation,6 while S1P3 receptor agonism has been
linked to acute toxicity and bradycardia in rodents.7 A
dose-dependent transient, asymptomic bradycardia has
been seen in clinical studies with 1,8 suggesting that
selecting against S1P3 may be desirable with second-
generation S1P receptor agonist immunosuppressants.
A previous report from these laboratories disclosed a
series of 1-benzyl-3-carboxyazetidine compounds (ex-
emplified by 3 and 4) as selective, orally bioavailable
S1P receptor agonists.9 While 3, 4, and many of their
analogues were found to be about 500- to 1000-fold
selective for S1P1 over S1P3, enhanced selectivity in this
class of compounds was often accompanied by modest,
yet significant, losses of S1P1 receptor affinity.
Departments of Medicinal Chemistry and Immunology &
Rheumatology Research, Merck Research Laboratories,
Rahway, New Jersey 07065, and Department of Preclinical
Safety Evaluation, Merck Research Laboratories,
West Point, Pennsylvania 19486
Received April 8, 2005
Abstract: A class of 3,5-diphenyl-1,2,4-oxadiazole based com-
pounds have been identified as potent sphingosine-1-phos-
phate-1 (S1P1) receptor agonists with minimal affinity for the
S1P2 and S1P3 receptor subtypes. Analogue 26 (S1P1 IC50
)
0.6 nM) has an excellent pharmacokinetics profile in the rat
and dog and is efficacious in a rat skin transplant model,
indicating that S1P3 receptor agonism is not a component of
immunosuppressive efficacy.
The advent of the novel immunosuppressant 2-amino-
2-(4-octylphenyl)ethylpropane-1,3-diol1 (1, FTY720) may
represent a new modality of immunosuppression and
Herein, we report the discovery of a class of potency-
enhanced S1P1 receptor agonists based on a 3,5-diphen-
yl-1,2,4-oxadiazole scaffold that exhibit exceptional
selectivity against S1P2 and S1P3 receptor subtypes.
Their syntheses, structure-activity relationships, and
the significance of their enhanced potency and selectiv-
ity as it impacts immunosuppressive efficacy and phar-
macology attributable to S1P3 receptor agonism are the
subjects of this communication.
* To whom correspondence should be addressed. Phone: (732) 594-
8319. Fax: (732) 594-8080. E-mail: zhen_li@merck.com.
† Department of Medicinal Chemistry.
‡ Current address: Millennium Pharmaceuticals Inc., 35 Landsowne
Street, Cambridge, MA 02139.
Current address: Abbott Laboratories, Building AP-10, 100 Abbott
Park Road, Abbott Park, IL 60064.
§ Department of Immunology & Rheumatology.
# Department of Preclinical Safety Evaluation.
| Current address: The Scripps Research Institute, ICND-118,
10550 N. Torrey Pines Road, La Jolla, CA 92037.
10.1021/jm0503244 CCC: $30.25 © 2005 American Chemical Society
Published on Web 09/10/2005

6170 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Letters
Table 1. S1P Receptor Affinities (IC50, nM)a and Rat
Scheme 1a
Pharmacokinetic Datab
a Reagents: (a) H2NNH2, EtOH, reflux (99%); (b) 4-(hydroxy-
methyl)benzoic acid, 2-chloro-1,3-dimethylimidazolinium chloride,
TEA, CH2Cl2 (50%); (c) (COCl)2, DMSO, DIEA, CH2Cl2, -78 °C to
room temp; (d) 3-carboxyazetidine, Na(CN)BH3, HOAc, MeOH
(yields, two steps: 8, 9, 50%; 12, 13, 50%; 16-26, 53%); (e)
ΗÃΝΗ2‚HCl NaHCO3, MeOH, reflux (10, 11, 95%; 14, 15, 99%);
(f) 4-(hydroxymethyl)benzoic acid, EDC, HOBT, CH3CN, reflux
(85%); (g) 4-(2-methylpropyl)benzoic acid, EDC, HOBT, CH3CN,
reflux (85%); (h) 2,2-dimethylpropylmagnesium bromide, Ni(dp-
pf)Cl2, ether, reflux; (i) cat. RuO2, NaIO4, EtOAc/H2O (52%, two
steps); (j) 4-(bromo)phenylboronic acid, cat. (R)-BINAP, cat.
Rh(acac)(C2H4)2, 9:1 v/v dioxane/H2O (54%, 91% ee); (k) [bis(2-
methoxyethyl)amino]sulfur trifluoride, BF3‚Et2O, toluene (65%);
(l) n-BuLi, CO2, THF/ether, -78 °C (75%).
a Displacement of [33P]-labeled sphingosine-1-phosphate (S1P)
by test compounds from human S1P receptors expressed on CHO
cell membranes. Data are reported as the mean of n ) 3
determinations. All compounds had S1P2 IC50 > 10 000 nM. SD
were generally (20% of the average. See ref 7a for assay protocol.
b 1 mg/kg iv. Units: Clp, mL min-1 kg-1, Vdss, L/kg, t1/2, h. Data
are reported as the average for n ) 2 iv. Compound plasma levels
for individual animals used to calculate PK parameters were with
(25% of average. See ref 19.
After in-house leads identified in a high-throughput
screen for S1P1 agonists10 led to the rational design of
3 and 4, approximately 1000 commercially available
compounds related to those leads were purchased and
tested for S1P receptor agonism activity. Compound 5,
chemistry used to prepare the benzoic acids needed for
targets 27 and 35. The synthesis of benzoic acid 20
(required for target 36) featured an asymmetric conju-
gate addition of 4-(bromo)phenylboronic acid to cyclo-
pentenone;14 analogous chemistry was used to prepare
the benzoic acids needed for 37.
Ligand competition studies between [33P]S1P and all
new compounds were carried out for each of the five
human S1P receptors stably expressed in Chinese
hamster ovary (CHO) cell membranes.4a S1P receptor
agonism by the test compounds was determined by
measurement of ligand-induced [35S]-5′-O-3-thiotriph-
osphate (GTPγS) binding.15 Some general features about
the data generated for the new compounds are note-
worthy (Table 1). While the S1P1 IC50 values for the new
compounds (9, 13, 21-37) spanned almost 100-fold, all
of them had minimal affinity for S1P2 and S1P3 recep-
tors. This absolute disconnection of S1P1 and S1P3 SARs
is in sharp contrast to what was seen previously for
other classes of S1P receptor agonists where these
receptor affinities were separable but effected by changes
in structure in similar ways.9,16 Most of the new
with a 3,5-diphenyl substituted oxadiazole moiety, was
identified in this set of analogues. Employing 5 as we
had our previous leads, we started the investigation of
3,5-diphenyl substituted oxadiazole based compounds,
which has led to the discovery of compound 26.
Representative syntheses are outlined in Scheme 1.
The key step in obtaining 9, 13, and 26 was the
formation of their respective appropriately substituted
oxadiazole heterocycles; this was efficiently realized
using chemistry based on established literature proce-
dures.11 Syntheses of 4-(cyclopropyl)-,12a 4-(cyclo-
butyl)-,12a 4-(cyclopentyl)-,12a and 4-(4,4-difluorocyclo-
hexyl)benzoic acid12b have appeared in the literature.
The preparation of benzoic acid 18, which featured a
nickel-catalyzed cross-coupling,13 is representative of the

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6171
Table 2. Minimum Single Dose (mg/kg, po) Required for
compounds showed a more modest 100-fold selectivity
against S1P4 while being nonselective in regard to S1P5.
Interestingly, compounds such as 26 were found to be
inactive in the GTPγS binding and ERK activation17
assays used to assess S1P4 functional activity which
indicates that the new compounds differ from com-
pounds such as 3 and 4 in that they are weak antago-
nists of the S1P4 receptor.
Maximal PBL Lowering Response 24 h Postdose
compd
mouse
rat
dog
1
3
26
1.0a
10a,b
10a
0.2a
3.0c
0.5a
0.1c
1.0c
0.5c
a Data generated 24 h after compound challenge in n ) 3
animals according to protocol described in ref 4a. b b.i.d. c Data
generated from PK/PD time-course studies in n ) 3 animals as
described in ref 9.
The S1P1 receptor data (Table 1) for the three 4-(tert-
butyl)phenyloxadiazole analogues 9, 13, and 21 indi-
cated a preference for the heterocycle of the last isomer.
There appears to be an optimal length and/or size of
the substituent of analogues of 21 at which S1P1 affinity
is maximized. Extending the carbon chain on the
pendent phenyl ring of analogues of 21 was found to be
S1P1 affinity-enhancing with isobutyl analogue (26)
having subnanomolar S1P1 IC50 values, but this had
limits because n-hexyl analogue 25 was significantly
less potent. The S1P1 data for cycloalkyl analogues 30-
33 and phenyl analogue 34 also support the notion that
an appropriately sized alkyl substituent affords maxi-
mal binding to S1P1. Fluorination of some selected alkyl
groups was found to be tolerated and in one case S1P1-
potency-enhancing (compare 35 vs 23). As previously
noted, none of these structural changes significantly
altered selection against S1P4 or S1P5 compared to that
seen for lead analogue 21.
tration of peripheral blood lymphocytes (PBLs), measure-
ment of PBL levels can be used as a surrogate marker
for efficacy amenable to the screening of multiple ana-
logues in vivo.18 The ability of these new compounds to
lower PBL counts after oral administration9 provided
another basis for analogue differentiation. While many
of the compounds in Table 1 were capable of eliciting a
maximal PBL lowering response 3 h after a sufficient
oral dose, only 26, 29, 32, 36, and 37 were capable of
doing so after a dose as low as 0.3 mpk po. While murine
pharmacokinetics were not determined as part of these
screening experiments, it is noteworthy that the most
potent of the new compounds in this assay were also
the ones with the best rat pharmacokinetics profiles.
The pharmacodynamic ED50 for 26 in the PBL lowering
screening assay was determined to be 0.03 mpk po,
making it comparable to 1 and approximately 10-fold
more potent than 3 or 4. The oral doses of 1, 3, and 26
required to elicit a maximum PBL lowering response
24 h postdose in the mouse, rat, and dog were also
determined (Table 2); PBL counts appeared to rebound
in all three species when plasma concentrations of 26
reached 25-50 nM, indicating that this compound was
at least 3- to 4-fold more potent than 3 in its ability to
drive the lowering of PBLs.21
It was possible to differentiate among the new com-
pounds based on their rat pharmacokinetics (PK) and
their ability to drive a pharmacodynamic (PD) response
characteristic of S1P1 receptor agonists, i.e., the dose
response of lowering peripheral blood lymphocyte
counts.4a,6,18 While all of the compounds for which rat
pharmacokinetics were determined19 were highly orally
bioavailable (>50%), many of the more interesting
simple alkyl and cycloalkyl analogues (e.g., 23, 32, 33)
were found to have high rates of plasma clearance in
relation to relatively modest steady-state volumes of
distribution. Isobutyl analogue 26 and phenyl analogue
34 were outliers in this respect with significantly longer
half-lives in the rat (26 t1/2 ) 8.5 h, 34 t1/2 ) 11 h). It
was proposed that metabolism of the terminal alkyl
groups of the higher clearance compounds might be
responsible in part for their poorer pharmacokinetics;
a subsequent metabolism and disposition experiment
in bile duct cannulated Sprague-Dawley rats with [14C]-
26 revealed that oxidation of the isobutyl group, the
phenyl ring to which it is attached, and the azetidine
nitrogen were major phase I metabolic processes for that
compound.20 The problem of high clearance could be
remedied with fluorination of the pendent alkyl groups;
analogues 35-37 were all found to have extended half-
lives compared to their des-fluoro counterparts. While
it is not clear why isobutyl analogue 26 is a better
pharmacokinetics player than many of its close ana-
logues, it is interesting to note that isopropoxy analogue
29 (which is isosteric to 26) also had an extended rat
half-life (t1/2 ) 7.1 h). Beagle dog pharmacokinetics9 for
selected compounds (23, 26, 29, 32, 33) were found to
parallel those in the rat, with 26 having a superior
On the basis of its S1P1 receptor agonist potency,
selectivity, pharmacokinetics/pharmacodynamic proper-
ties, and the ready availability of the intermediates, 26
was subjected to more in-depth characterizations.
The ability of 1 to prolong allograft survival in a var-
iety of preclinical species and to synergize with agents
that effect lymphocyte proliferation and IL-2 production
(such as CsA and FK-506) is well-established.22 To dem-
onstrate that an S1P1 agonist with attenuated affinity
for S1P3 receptors could be expected to maintain those
properties of 1, a rat skin allograft experiment based
on a previously published protocol23 was conducted.
Compounds 26 (5.0 mpk po/day, 28 days, n ) 14 rats)
and 1 (1 mpk po/day, 28 days, n ) 14 rats) were
independently combined with a subtherapeutic dose of
CsA (1 mpk/day, delivered intraperitoneally till graft
death) and tested in Lewis rats that received skin grafts
from DA rats. The median graft survival times were 22
days in the 26 + CsA arm (100% of grafts were rejected
by day 43) and 13 days in the 1 + CsA arm (100% of
grafts were rejected by day 34). Rats treated with
vehicle or CsA alone had 100% graft death between days
10 and 12, respectively. Exposure to CsA was compa-
rable in all of the cohorts to which it was administered.
These results indicate that S1P receptor agonists that
select against the S1P3 subtype should be expected to
be efficacious as immunosuppressive agents. The con-
sequences for efficacy of selecting against the S1P4 and/
or S1P5 receptor subtypes remain to be established.
profile (Clp ) 3.3 mL min-1kg-1, Vdss ) 2.5 L/kg, t1/2
)
10 h, % F ) 66 after 1.0 mpk po and 0.5 mpk iv doses).
Since the immunosuppressive efficacy of 1 has been
proposed to arise from its ability to promote the seques-

6172 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Letters
Cardiovascular evaluations of the new compounds in
the previously described7a conscious rat model were hin-
dered by our inability to administer compounds at doses
sufficient to elicit responses attributable S1P3 agon-
ism.24 Fortunately, a determination of compound effects
on airways resistance in the rat appears to provide an
attractive means for demonstrating the undesirability
of S1P3 agonism. In addition to modulating lymphocyte
trafficking, it was recently demonstrated that S1P can
stimulate the contraction of human airway smooth mus-
cle in vitro.25 While this finding implies that S1P has a
role in airway tone, it was uncertain whether S1P re-
ceptor activation would augment airway resistance in
a whole animal model. To assess whether S1P receptors
can alter airway resistance, the respiratory effects of
1, 3, and 26 were evaluated in anesthetized rats. In this
study, airway resistance was measured directly using
a computer-controlled small animal ventilator to mea-
sure drug-induced changes in respiratory mechanics.26
In this model, intravenous infusion of the muscarinic
agonist methacholine (30 µg kg-1 min-1 × 30 min), a
known bronchconstrictor, caused a significant increase
in airway resistance (152 ( 6%) in anesthetized rats.
Administration of 1 and 3 at 10 mpk iv infused over 30
min induced bronchoconstriction and elevated airway
resistance by 344 ( 33% and 140 ( 5% compared to
baseline. Infusion of 26 was devoid of any effect on air-
way resistance and was similar to vehicle treatment
(104 ( 1%). These findings suggest that agonists with
high to moderate affinity for S1P3 receptors, e.g, 1 and
3, can produce bronchoconstriction in the rat and sug-
gest that airways smooth muscle contraction can be me-
diated by S1P3 receptors. This is supported by the obser-
vation that a highly selective S1P1 agonist with minimal
affinity for S1P3 receptors, e.g, 26, lacked the ability to
stimulate airway smooth muscle contraction in vivo.
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exemplified by 26 with high selectivity against the S1P2
and S1P3 receptor subtypes have been discovered. While
it has been previously shown that lymphocyte traffick-
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Acknowledgment. We are grateful to Drs. Larry
Colwell and William Feeney for coordinating the rat and
dog pharmacokinetics studies, Dr. Bernard Choi and
Ms. Falguni Patel for HRMS determinations, and Ms.
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