Highly selective and potent agonists of sphingosine-1-phosphate 1 (S1P1) receptor

Article abstract of DOI:10.1016/j.bmcl.2006.04.064

Novel series of sphingosine-1-phosphate (S1P) receptor agonists were developed through a systematic SAR aimed to achieve high selectivity for a single member of the S1P family of receptors, S1P₁. The optimized structure represents a highly S1P<

Full text of DOI:10.1016/j.bmcl.2006.04.064

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3684–3687
Highly selective and potent agonists of sphingosine-1-phosphate 1
(S1P1) receptor
Petr Vachal,a,* Leslie M. Toth,a Jeffrey J. Hale,a Lin Yan,a Sander G. Mills,a
Gary L. Chrebet,b Carol A. Koehane,b Richard Hajdu,b James A. Milligan,b
Mark J. Rosenbachb and Suzanne Mandalab
aDepartment of Medicinal Chemistry, Merck & Co., Inc., PO Box 2000, Rahway, NJ 07065, USA
bDepartment of Immunology and Rheumatology, Merck & Co., Inc., PO Box 2000, Rahway, NJ 07065, USA
Received 8 March 2006; accepted 21 April 2006
Available online 6 May 2006
Abstract—Novel series of sphingosine-1-phosphate (S1P) receptor agonists were developed through a systematic SAR aimed to
achieve high selectivity for a single member of the S1P family of receptors, S1P1. The optimized structure represents a highly
S1P1-selective and efficacious agonist: S1P1/S1P2, S1P1/S1P3, S1P1/S1P4 > 10,000-fold, S1P1/S1P5 > 600-fold, while EC50 (S1P1)
<0.2 nM. In vivo experiments are consistent with S1P1 receptor agonism alone being sufficient for achieving desired lymphocyte-
lowering effect.
Ó 2006 Elsevier Ltd. All rights reserved.
Sphingosine-1-phosphate (S1P)1 induces a range of cel-
lular responses by its interaction with the S1P family
of G-protein coupled receptors (GPCRs). A total of five
S1P receptors are known: S1P1–S1P5. The in vivo immu-
nosuppressive efficacy of a non-selective S1P receptor
agonist has been evidenced by the numerous preclinical
and clinical studies of FTY7202,3 (Novartis), a prodrug
for its monophosphate ester.4 Recent studies have dem-
onstrated that S1P receptor agonists affect lymphocyte
trafficking5 and that an interaction with S1P1 receptors
on lymphocytes drives the observed pharmacodynam-
ics.6 S1P3 receptor activity has been linked to acute tox-
icity and bradycardia in rodents.7 As no highly potent
and selective agonists have been reported to this date,8
definitive evidence has not yet been provided as to
whether a selective interaction with S1P1 receptors
would be sufficient for immunosuppressive efficacy. We
present here the design and SAR study leading to the
preparation of highly S1P1-selective agonists.
have good rat and dog pharmacokinetic profiles.5i,9 (4-
(1,2,4-Oxadiazol-3-yl)-3-methyl)phenylpropionic acids
1a–1c have been shown to be orally bioavailable potent
S1P1 receptor agonists that select against S1P2 and S1P4
subtypes.5b,5e–5g For the latter, the left-hand side aromatic
substituent can influence selectivity against S1P5 (Table
1).10 Based on these observations, we proposed to test a
new structural class of compounds with the SAR focused
on both S1P1 receptor efficacy and selectivity (Fig. 1).
In order to generate a diverse SAR dataset with respect
to the central heterocycle X (Fig. 1), we reasoned that
the core of the proposed series may be synthesized in a
three-step synthetic sequence consisting of two palladi-
um(0)-catalyzed cross-coupling reactions (Suzuki,
Negishi, or Stille couplings) and a bromination. This
hypothesis led to the development of a novel general
methodology yielding a variety of 2,5-diarylheteropenta-
lenes in a significantly more convergent manner com-
pared to previously reported alternatives (Scheme 1).
We believe that this synthetic approach represents the
most efficient route to a diverse array of 2,5-disubstitut-
ed heterocycles yet reported.11–13
N-(4-(1,3,4-Oxadiazole)benzyl)azetidine-3-carboxylic and
N-(4-(1,2,4-oxadiazole)benzyl)azetidine-3-carboxylic acids
have been previously reported to be potent S1P1 receptor
agonists that select against S1P2 and S1P3 subtypes and
A direct comparison of several heterocyclic analogs
sharing identical aromatic 2,5-disubstitution validated
the basis of our proposal: S1P1/S1P3 selectivity varies
profoundly as a function of the central heterocycle
(Table 1). Superior selectivity against the S1P3 receptor
Keywords: Sphingosine-1-phosphate; S1P; Immunosuppressant;
Lymphocyte-lowering; Agonist; Heteropentalene.
*
Corresponding author. Tel.: +1 732 594 6624; fax: +1 732 594
2210; e-mail: petr_vachal@merck.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.04.064

P. Vachal et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3684–3687
3685
Table 1. S1P receptor agonism as a function of central heterocycle X
and left-hand side aromatic substituent Ar1 in 3-(4-heterocyclyl-3-
methyl-phenyl)propionic acid series (1c–g)
As previously disclosed series of S1P agonists have gen-
erally not selected against S1P5 subtype, we focused on
achieving a good S1P1/S1P5 selectivity, while maintain-
ing strong affinity for S1P1 receptor by optimizing lead
1e. In due course, we benefited from the parallel agree-
ment of trends in S1P1 receptor efficacy invoked by cor-
responding members of two simultaneously optimized
series: 1,2,4-oxadiazoles (represented by 1c) and 1,3,4-
thiadiazoles (represented by 1e).5d–g,14 The SAR of the
two series suggested that the superior substitution pat-
tern for Ar1 was 3,4-disubstitution.15 S1P1 efficacy in
1,3,4-thiadiazole series benefited from the strongly
electron-withdrawing nature of the 3-substituent, S1P1/
S1P5 selectivity was mostly dependent on the nature of
the 4-substituent. Generally, an electron-donating ether
or alkyl substituent in the 4-position provided the high-
est selectivity against S1P5 receptor. A detailed SAR of
Me
X
Ar1
CO2H
Compound
X
Ar1
EC50 (nM)a,b or %
inhibition @ 10 lM
S1P1 S1P3 S1P5
F3C
N
O
O
O
N
N
O
S
1a
1b
1c
1d
1e
1f
0.10
0.15
0.73
19
110
940
950
82%
36%
13
iPrO
Cl
N
N
73
iPrO
Cl
N
N
950
44%
1300
0%
6%
iPrO
Cl
N
N
N
N
N
N
N
Table 2. Parallel of S1P1 receptor agonism in 1,3,4-thiadiazole and
1,2,4-oxadiazole series expedited identification of the optimal substi-
tution pattern of the 1,3,4-thiadiazole series; SAR optimization of the
4-substituent in 3-cyanophenyl-1,3,4-thiadiazole series (2a-v) for S1P1
receptor efficacy and S1P5/S1P1 selectivity
iPrO
Cl
O
N
2.1
iPrO
Cl
S
Me
Me
N
N
O N
Ar1
Ar1
1000 8%
S
N
CO2H
CO2H
iPrO
Cl
N
N
1,3,4-thiadiazole series
1,2,4-oxadiazole series
SAR of over 50 compounds
1g
30%
0%
iPrO
Me
N
N
a For a summary of biological assays used in this report, see Ref. 10;
SD were generally 20% of the average.
b EC50 and IC50 values for S1P2 and S1P4 receptors, respectively,
exceeded the S1P1 receptor EC50 by more than 1000-fold.
NC
R
S
CO2H
3,4-disubstitution superior
a
b
Compound, R
EC50
Selectivity S1P5/S1P1
2a, H
2b, MeO
>1000
97
2.1
N/Ac
>100
480
110
210
320
200
230
120
170
18
Me
Left-Hand Side
Aromatic Substituent
Ar1
CO2H
Central
Heterocycle
X
2c, EtO
2d, n-PrO
3.4
2e, i-PrO
2f, i-Bu
2g, i-BuO
0.20
0.16
2.8
Figure 1. Design of the structural series reported in this disclosure.
2h, cyc-PrCH2O
2i, sec-BuO
2j, cyc-BuO
2k, F
0.61
0.28
0.79
570
9.5
Br2, AcONa
Y
Z
Y
Z
Z
Pd(Ph3P)4
AcOH
Ar1I
+
5 mol% Ar1
[M]
X
X
X, Y, Z = CH, O, S, or N
[M] = SnBu3, ZnBr, B(OH)2
2l, FCH2CH2O
2m, CF2HCH2O
2n, CF3CH2O
2o, CF3(CH2)2O
2p, (CF3)2CHO
2q, HO2CCH2O
2r, NCCH2O
2s, F3CH(Me)O
2t, (FCH2)2CHO
2u, AcO
>100
230
400
30
Furans
Thiophenes
Pyrroles
1.1
0.6
1,3-Oxazoles
Ar2B(OH)2
Pd(Ph3P)4
Y
7.7
38
1,3-Thiazoles
1,3,4-Oxadiazoles
1,3,4-Thiadiazoles
(> 20 examples)
Y
Z
Ar1
Ar2
2
28
Ar1
Br
X
X
5 mol%
360
45
3 steps
31 - 67% overall yield
220
93
0.19
0.23
94
Scheme 1. General synthesis of 2,5-diarylheteropentalenes served for
the preparation of many of the compounds reported in this
disclosure.11
390
110
110
2v, 2-thienyl
1.9
a EC50 (nM) for S1P1 receptor; for a summary of biological assays used
in this report, see Ref. 10; SD were generally 20% of the average.
b EC50 and IC50 values for S1P2 and S1P4 receptors, respectively,
exceeded the S1P1 receptor EC50 by more than 1000-fold. S1P5/S1P1
is defined as the ratio of the respective EC50 values.
observed for 1,3,4-thiadiazole 1e relative to its deriva-
tives is particularly important given the association of
S1P3 agonism and acute toxicity and bradycardia in
rodents.7
c S1P5/S1P1 not calculated due to low S1P1 receptor efficacy.

3686
P. Vachal et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3684–3687
Table 3. S1P receptor agonism as function of central heterocycle X
in 3-cyano-4-isopropyloxyphenyl left-hand side series (1a, 2e, and
3a–n)
2-((3-cyano-4-substituted)-phenyl)-1,3,4-thiadiazoles opti-
mizing the 4-position for the highest S1P1 receptor efficacy
and S1P1/S1P5 selectivity is summarized in Table 2.16 The
most S1P1/S1P5 selective (>200-fold) as well as highly
S1P1 efficacious (EC50 < 1 nM) derivatives include 2e, 2f,
2h, 2n, and 2t.
Me
NC
X
CO2H
iPrO
Compound
X
EC50 (nM)a,b
S1P3
Having identified several desirable left-hand side aro-
matic substituents, we conducted a detailed SAR of
the central heterocycle, while maintaining both the left-
and right-hand side aromatic substituents unchanged.17
Our three-step synthetic sequence (Scheme 1) provided
facile access to the core of many compounds listed in
Table 3.18 While many of the heterocyclic derivatives
proved to be efficacious S1P1 receptor agonists, only a
handful of these concurrently exhibited a high degree
of selectivity for S1P1 receptor against all other members
of the S1P family: thiadiazole 2e, oxadiazole 3a, thio-
phene 3b, thiazole 3f, and tetrazole 3i (Table 3). Among
these, thiazole 3f was found to be the most effective to
maximally drive the lymphocyte-lowering response
(MOSL) inducing the strongest lymphopenia after oral
administration to mice.10,19
S1P1
0.08
S1P5
6.5
N
O
N
N
1a
2e
3a
3b
3c
3d
3e
3f
1144
N
N
0.20
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
42
S
N
1.1
460
O
0.93
220
S
>10,000
>10,000
14
>10,000
>10,000
200
O
In conclusion, a systematic SAR analysis has identified
selective, highly potent, small molecule S1P1 receptor
agonists (Table 3). Optimized structures are exemplified
by compound 3f with an in vitro S1P1 receptor efficacy
EC50 = 0.18 nM, greater than 10,000-fold selectivity
against S1P2, S1P3, and S1P4 receptors and greater than
600-fold selectivity against S1P5 receptor. After oral
administration of a 0.3 mpk dose of 3f to mice, it was
found to maximally drive the lymphocyte-lowering re-
sponse previously seen with other S1P receptor ago-
nists.10 These results are consistent with the notion
that selective agonism of S1P1 receptor is sufficient for
achieving the desired degree of lymphocyte-lowering.
While agonist 3f exhibited good oral bioavailability in
the rat (F = 39%), its pharmacokinetic properties in that
species remained suboptimal (t1/2 < 1 h). The optimiza-
tion of 3f toward analogs with more desirable pharma-
cokinetic profiles will be a subject of our future
disclosures.
N
Me
N
S
N
S
0.18
120
Me
N
3g
3h
3i
>3600
1.6
>10,000
43
S
N
N
N
N
N
N
0.98
150
N
N
N
3j
5.1
>10,000
3200
O
References and notes
S
N
3k
3l
290
1. For recent reviews on S1P receptors, see: (a) Cyster, J. G.
Ann. Rev. Immun. 2005, 23, 127; (b) Rosen, H.; Goetzl, E.
J. Nat. Rev. Immun. 2005, 5, 560.
2. FTY720 is currently in Phase III clinical trials for organ
transplantation and in Phase II for the treatment of
multiple sclerosis; Novartis Press Release Jun-21-2005,
Basel, Switzerland.
3. For recent reviews on FTY720, see: (a) Brinkmann, V.;
Lynch, K. R. Curr. Opin. Immun. 2002, 14, 569; (b)
Brinkmann, V.; Cyster, J. G.; Hla, T. Am. J. Transpl.
2004, 4, 1019.
4. 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. Science 2002, 296,
346.
S
N
1200
>10,000
380
3200
3m
3n
>10,000
>10,000
N
Me
N
N
N
H
a For a summary of biological assays used in this report, see Ref. 10;
SD were generally 20% of the average.
b EC50 and IC50 values for S1P2 and S1P4 receptors, respectively,
exceeded the S1P1 receptor EC50 by more then 1000-fold.

P. Vachal et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3684–3687
3687
5. (a) Hale, J. J.; Neway, W.; Mills, S. G.; Hajdu, R.;
Keohane, C. A.; Rosenbach, M.; Milligan, J.; Shei, G. J.;
Chrebet, G.; Bergstrom, J.; Card, D.; Koo, G. C.;
Koprak, S. L.; Jackson, J. J.; Rosen, H.; Mandala, S.
Bioorg. Med. Chem. Lett. 2004, 14, 3351; (b) Colandrea,
V. J.; Doherty, G. A.; Hale, J. J.; Lynch, C.; Mills, S. G.;
Neway, W. E.; Toth, L. PCT Int. Appl. 2004, WO
2004103279, A2 20041202; (c) 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. J. Med.
Chem. 2004, 47, 6662; (d) Hale, J. J. In 229th ACS
National Meeting, Mar-13-2005, San Diego, CA; (e)
Doherty, G. A.; Hale, J. J.; Legiec, I. E.; Lynch, C. L.;
Toth, L. M. PCT Int. Appl. 2005, WO 2005032465, A2
20050414; (f) Yan, L.; Huo, P.; Doherty, G.; Toth, L.;
Hale, J.; Mills, S.; Hajdu, R.; Keohane, C.; Rosenbach,
M.; Milligan, J.; Shei, G.-J.; Chrebet, G.; Bergstrom, J.;
Card, D.; Quackenbush, E.; Wickham, A.; Mandala, S. In
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2005, Antwerp, Belgium; (g) Colandrea, V. J.; Doherty, G.
A.; Hale, J. J.; Huo, P.; Legiec, I. E.; Toth, L.; Vachal, P.;
Yan, L. PCT Int. Appl. 2005, WO 2005058848, A1
20050630; (h) Vachal, P.; Toth, L.; Bugianesi, R.; Hale,
J. J.; Neway, W.; Yan, L.; Huo, P.; Mills, S. G.; Chrebet,
G.; Keohane, C. A.; Hajdu, R.; Rosenbach, M. J.;
Milligan, J. A.; Mandala, S. In Gordon Research Confer-
ence on Natural Products, Jul-24-2005, Tilton, NH; (i)
Zhen, L.; Weirong, C.; Hale, J. J.; Lynch, C.; Mills, S. G.;
Hajdu, R.; Keohane, C. A.; Rosenbach, M.; Milligan, J.;
Shei, G. J.; Chrebet, G.; Parent, S. A.; Bergstrom, J.;
Card, D.; Forrest, M.; Quackenbush, E. J.; Wickham, A.;
Vargas, H.; Evans, R. E.; Rosen, H.; Mandala, S. J. Med.
Chem. 2005, 48, 6169.
6. (a) Matloublan, M.; Lo, C. G; Cinamon, G.; Lesneski, M.
J.; Xu, Y.; Brinkmann, V.; Allende, M. L.; Proia, R. L.;
Cyster, J. G. Nature 2004, 427, 355; (b) Allande, M. L.;
Dreier, J. L.; Mandala, S.; Proia, R. L. J. Biol. Chem.
2004, 279, 15396.
7. Germana, S. M.; Liao, J.; Jo, E.; Alfonso, C.; Ahn, M.-Y.;
Peterson, M. S.; Webb, B.; Lefebvre, S.; Chun, J.; Gray,
N.; Rosen, H. J. Biol. Chem. 2004, 279, 13839.
8. For the most active and selective S1P1 receptor agonist
reported thus far, see Ref. 7 and footnote 2 therein.
9. A limited prior SAR of a few additional N-(4-(hetero-
cyclyl)benzyl)azetidine-3-carboxylic acids have suggested
that variation of the central heterocycle may be tolerated
without compromising the affinity for S1P1 receptors:
Hale, J. J.; Bugianesi, R.; Neway, W. Unpublished results.
10. Summary of the primary biological assay used in this
report: In vitro EC50: agonism of GPCR receptors:
induced [35S]GTPcS binding by test compounds (see Ref.
4 for details); In vivo Pharmacokinetics in the rat.
Pharmacodynamics: induction of peripheral lymphocyte
lowering (MOSL; see Ref. 5a for details).
was degassed with a steady stream of argon for 10 min.
Solid Pd(Ph3P)4 (0.1 mmol) was added and the mixture
was degassed with argon for 2 min after which it was
heated under argon to 85 °C for 3 h. The reaction mixture
was combined with 1 M HCl (100 mL) and ethyl acetate
(200 mL). The organic layer was separated, washed
sequentially with 1 M HCl (50 mL) and brine (50 mL),
and dried over sodium sulfate. Desired product was
obtained by column chromatography. See Ref. 11 for
details.
13. A representative procedure for selective bromination of 2-
arylheteropentalenes: to a stirred homogeneous solution
of 2-arylheteropentalene (5.0 mmol) and sodium acetate
(10 mmol) in acetic acid (25 mL), bromine (5.0 mmol) was
added dropwise via syringe at room temperature over
30 min. The reaction progress was monitored by TLC or
LC–MS analyses: upon completion, the reaction mixture
was combined with 1 M NaOH (250 mL) and ethyl acetate
(250 mL), the organic layer separated, washed sequentially
with 1 M NaOH (100 mL) and brine (100 mL), and dried
over sodium sulfate. Desired product was obtained by
column chromatography. See Ref.11 for details.
14. Parallel nature of the two series is exemplified in Table 1
(1c vs. 1e) and Table 3 (1a vs. 2e) of this report. Additional
examples of similarities between corresponding 1,2,4-
oxadiazole and 1,3,4-thiadiazole efficacy for S1P1 receptor
include (left-hand side aryl residues listed): 4-isopropyl-
oxy-3-(trifluoromethyl)phenyl (EC50 = 0.09 vs. 0.13 nM)
and 4-cyclohexylphenyl (EC50 = 7.1 vs. 11 nM), respec-
tively. For a full scope of the analogy, see Ref. 5g.
15. Key role of 3,4-disubstitution for S1P1 receptor efficacy is
in a good agreement with trends observed in several
previously reported series of S1P agonists; see Ref. 5 for
details.
16. Only EC50 values for S1P1 receptor are listed in Table 3 as
the efficacy for this receptor and selectivity against S1P5
receptor were the primary objectives of the SAR described
therein. The EC50 values for S1P2–S1P4 receptors gener-
ally exceeded the S1P1 EC50 by more than 1000-fold.
17. The primary reason for selecting 2e among the potential
candidates for further optimization was the in vivo efficacy
after oral administration to mice: 2e was found to be most
effective agonist to maximally drive the lymphocyte-
lowering response at 0.3 mpk dose; see Ref. 10 for details.
18. The following scheme represents a typical overall synthetic
route to compounds in Table 3:
Me
Y
Z
Y
Z
Scheme 1, ref. 11
3 steps
NC
F
iPrONa, THF
Br
Me
[M]
X
X
X, Y, Z = CH, O, S, or N
[M] = SnBu3, ZnBr, B(OH)2
Y
Z
NC
iPrO
Me
EtO2C
Y
Z
ZnCl
NC
iPrO
X
CO2R
Pd(P-t-Bu3)2
X
Br
LiOH
R = Et
R = H
11. This synthetic strategy was previously communicated:
Vachal, P.; Toth, L. Tet. Lett. 2004, 45, 7157.
12. A representative procedure for the Suzuki coupling: a
heterogeneous mixture of 5-bromo-2-arylheteropentalane
(1.0 mmol), DMF (20 mL), 1 M aqueous solution of
sodium carbonate (5 mL), and boronic acid (1.1 mmol)
19. While 3 mpk of each of 2e, 3a, 3b, and 3i was needed
to maximally drive the lymphocyte-lowering after oral
administration to mice, 0.3 mpk dose of 3f was
sufficient to achieve the same effect; see Ref. 10 for
details.