2,5-Disubstituted pyrrolidine carboxylates as potent, orally active sphingosine-1-phosphate (S1P) receptor agonists

Article abstract of DOI:10.1016/j.bmcl.2006.03.038

A series of 2,5-cis-disubstituted pyrrolidines were synthesized and evaluated as S1P receptor agonists. Compounds 15-21 were identified with good selectivity over S1P₃ which lowered circulating lymphocytes after oral administration in mice.

Full text of DOI:10.1016/j.bmcl.2006.03.038

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2905–2908
2,5-Disubstituted pyrrolidine carboxylates as potent, orally active
sphingosine-1-phosphate (S1P) receptor agonists
Vincent J. Colandrea,a,* Irene E. Legiec,a Pei Huo,a Lin Yan,a Jeffrey J. Hale,a
Sander G. Mills,a James Bergstrom,b Deborah Card,b Gary Chebret,b Richard Hajdu,b
Carol Ann Keohane,b James A. Milligan,b Mark J. Rosenbach,b
Gan-Ju Sheib and Suzanne M. Mandalab
aDepartment of Medical Chemistry, Merck Research Laboratories, Rahway, NJ 07065, USA
bDepartment of Immunology and Rheumatology Research, Merck Research Laboratories, Rahway, NJ 07065, USA
Received 3 February 2006; revised 28 February 2006; accepted 1 March 2006
Available online 31 March 2006
Abstract—A series of 2,5-cis-disubstituted pyrrolidines were synthesized and evaluated as S1P receptor agonists. Compounds 15–21
were identified with good selectivity over S1P3 which lowered circulating lymphocytes after oral administration in mice.
ꢀ 2006 Published by Elsevier Ltd.
The sphingosine-1-phosphate-1 (S1P1) receptor has
recently emerged as a novel molecular target for immuno-
suppression.1 Systemic administration of S1P agonists
results in the sequestration of peripheral blood lympho-
cytes (PBLs) in secondary lymphoid organs, which
prevents their access to transplanted or non-lymphoid
tissues.2 This pharmacodynamic phenomenon is puta-
tively responsible for the immunosuppressive efficacy of
this class of compounds.3
tuted pyrrolidines was designed, starting with ( )-pyr-
rolglutamic acid 3. Schemes 1–3 illustrate our synthetic
approach. Sequential protection of ( )-3 under standard
conditions gave the N-tert-butoxycarbamoyl methyl es-
ter ( )-4.7 Regioselective addition to the amide carbonyl
with 4-cyanophenylmagnesium chloride gave ketone
( )-5.8 Treatment of ( )-5 with trifluoroacetic acid
effected ring closure to the corresponding pyrroline,
which was subsequently reduced with sodium cyano-
borohydride to provide the diastereomeric ( )-cis- and
( )-trans-pyrrolidines ( )-6a,b. These diastereomers
were separated by flash chromatography and assign-
ment of their relative stereochemistries was secured by
ID nOe experiments.9 Protection of the pyrrolidine
nitrogen afforded nitriles ( )-7a,b.
Work from these laboratories has shown that the 2,5-di-
substituted pyrrolidine ( )-1 and diaryl-1,2,4-oxadiazole
2 are potent agonists of S1P receptors.4,5 In addition,
compound 2 and its analogs were found to have excep-
tional selectivity against S1P3, a receptor subtype that
mediates acute cardiovascular toxicity in rodents.6
Based on these results, we sought to combine the salient
features of the oxadiazole-based lipophilic domain of
compounds like 2 and the pyrrolidine scaffold in ( )-1
with the aim of affording potent, selective, and orally ac-
tive S1P1 agonists (Fig. 1).
PO3H2
N
H
n-C9H19
(+/-)-1
In order to modify both the 2- and 5-positions of the
pyrrolidine scaffold, a flexible synthesis of these disubsti-
N
N
Keywords: Pyrrolidines; Oxadiazole; S1P receptor; Immuno-
suppressants.
CO2H
O N
2
*
Corresponding author. Tel.: +1 732 594 1669; fax: +1 732 594
5966; e-mail: vince_colandrea@merck.com
Figure 1.
0960-894X/$ - see front matter ꢀ 2006 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2006.03.038

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V. J. Colandrea et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2905–2908
c
a,b
a,b
c,d,e
(+/-)-7a (2,5-cis)
(+/-)-7b (2,5-trans)
O
CO2H
O
CO2Me
d,e
CHO
N
N
N
H
BOC
BOC
NC
(+/-)-12a (2,5-cis)
(+/-)-12b (2,5-trans)
(+/-)-3
(+/-)-4
O
NHBOC
CO2Me
f,g,h
CO2Et
(+/-)-5
NC
H2N
N
BOC
(+/-)-13a (2,5-cis)
(+/-)-13b (2,5-trans)
N
HO
nOE
nOE
H
CO2H
H
H
H
N
H
N
+
CO2Me
CO2Me
N
N
R
R
NC
NC
O N
(+/-)-14a (2,5-cis)
(+/-)-14b (2,5-trans)
(+/-)-6a, R = H
(+/-)-6b, R = H
f
f
Scheme 4. Reagents and conditions: (a) LiBH4, THF; (b) (COC1)2,
DMSO, then Et3N, ꢀ78 ꢁC to rt; (c) ethyl(triphenylphosphoranyli-
dine) acetate, PhCH3; (d) H2, 10% Pd–C, CH3OH; (e) NH2OH, Et3N,
CH3OH (( )-7a: 11%, ( )-7b 16%, four steps); (f) EDC, 4-(2-
methylpropyl)-phenylbenzoic acid, CH3CN, rt then 120 ꢁC, 15 h; (g)
TFA, CH2Cl2; (h) NaOH, CH3OH (( )-14a: 68%, ( )-14: 22%, three
steps).
(+/-)-7a, R = BOC
(+/-)-7b, R = BOC
Scheme 1. Reagents and conditions: (a) Amberlyst-15, CH3OH, 50 ꢁC;
(b) Boc2O, Et3N, DMAP (78%, two steps); (c) 4-cyanophenylmagne-
sium chloride, ꢀ40 ꢁC; (d) TFA, CH2Cl2; (e) NaBH3CN, HCl,
CH3OH (6a: 38%, 6b: 24%, three steps); (f) Boc2O, CH2CI2 (7a:
96%, 7b: 80%).
( )-7b with hydroxylamine gave the amidoximes
( )-8a and ( )-8b. These intermediates were O-acylated
and thermally cyclized and dehydrated to afford the
corresponding 1,2,4-oxadiazoles. Deprotection of the
N-tert-butoxycarbamate and methyl ester moieties
afforded compounds ( )-9a and ( )-9b.
CO2Me
a
H2N
HO
N
(+/-)-7a (2,5-cis)
(+/-)-7b (2,5-trans)
BOC
(+/-)-8a (2,5-cis)
(+/-)-8b (2,5-trans)
N
b,c,d
CO2H
N
H
N
The homologation of nitriles ( )-7a and ( )-7b was
accomplished through an Arndt-Eistert sequence10 to
give esters ( )-10a and ( )-10b (Scheme 3). Installation
of the oxadiazole and deprotection vide infra furnished
the desired b-amino acids ( )-11a and ( )-11b.
O N
(+/-)-9a (2,5-cis)
(+/-)-9b (2,5-trans)
Scheme 2. Reagents and conditions: (a) NH2OH, Et3N, CH3OH (( )-
7a: 93%, ( )-7b 73%); (b) EDC, 4-(2-methylpropyl)phenylbenzoic acid,
CH3CN, rt then 120 ꢁC, 15 h; (c) TFA, CH2Cl2; (d) NaOH, CH3OH
(( )-9a: 51%, ( )-9b: 34%, three steps).
Preparation of the c-amino acids ( )-14a and ( )-14b is
outlined in Scheme 4. Selective reduction of the esters of
( )-7a and ( )-7b with lithium borohydride11 gave the
corresponding alcohols, which were oxidized using the
Swern protocol12 to furnish aldehydes ( )-12a and ( )-
12b. Reaction with ethyl(triphenylphosphoranylidine)
acetate, followed by hydrogenation and reaction with
hydroxylamine, gave amidoximes ( )-13a and ( )-13b.
Once again, oxadiazole formation followed by sequen-
tial deprotection afforded compounds ( )-14a and
( )-14b.
CO2Me
d,e,f,g
a,b,c
N
(+/-)-7a (2,5-cis)
(+/-)-7b (2,5-trans)
BOC
NC
(+/-)-10a (2,5-cis)
(+/-)-10b (2,5-trans)
CO2H
N
H
N
(+/-)-11a (2,5-cis)
(+/-)-11b (2,5-trans)
O N
Binding affinities for new compounds were evaluated for
each of the five known sphingosine-1-phosphate recep-
tors (S1P1–5) in radioligand competitive binding assays
using [33P]S1P expressed in Chinese hamster ovary
(CHO) cell membranes.1 S1P receptor agonism was
determined by measurement of ligand-induced [35S]-50-
O-3-thiotriphosphate (GTPcS) binding. All new
compounds were found to be agonists of the SlP1,3,5
receptors and to have minimal affinity for the S1P2
receptor subtype. Values for binding (IC50) and func-
tional (EC50) assays were in agreement to a factor of
4, thus only IC50 values for S1P1,3–5 receptors will be dis-
played for new compounds (see Tables 1 and 2). The
ability of selected compounds to lower circulating PBLs
Scheme 3. Reagents and conditions: (a) LiOH, THF/CH3OH/H2O; (b)
i-BuOCOCl, Et3N, then CH2N2; (c) AgOBz, Et3N, CH3OH (( )-10a:
52%, ( )-10b: 14%, three steps); (d) NH2OH, Et3N, CH3OH; (e) EDC,
4-(2-methylpropyl)-phenylbenzoic acid, CH3CN, rt then 120 ꢁC, 15 h;
(f) TFA, CH2Cl2; (g) NaOH, CH3OH (( )-11a: 45%, ( )-11b: 36%,
four steps).
Nitriles ( )-7a,b were valuable intermediates for the syn-
thesis of the pyrrolidine carboxylate homologs ( )-9a,
9b, 11a, 11b, 14a, and 14b (Schemes 2–4). Preparation
of the a-amino acids ( )-9a and ( )-9b is outlined in
Scheme 2. Independent treatment of nitriles ( )-7a and

V. J. Colandrea et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2905–2908
Table 1. S1P receptor affinities of carboxylate homologs
2907
CO2H
n
N
H
N
(+/-)
O
N
IC50 (nM)
Compound
n
Stereo
IC50 (nM)
S1P1
S1P3
S1P4
S1P5
9a
9b
0
0
1
1
2
2
cis
75
17
>10,000
10,000
1000
300
1280
180
200
100
30
trans
cis
11a
11b
14a
14b
3.3
8.0
4.5
5.7
>10,000
>10,000
>10,000
6700
1000
700
trans
cis
540
630
trans
80
Compounds ( )-9–14 were >10 lM for the S1P2 receptor.
Table 2. Binding affinities of compounds 15–21
CO2H
N
H
N
Ar
(+/-)
O
N
Compound
Ar
IC50 (nM)
S1P1
S1P3
1700
S1P4
350
S1P5
20
15
16
17
18
19
0.7
2.7
2.0
0.8
1.2
>10,000
7600
1400
2100
720
60
30
16
13
F
F
H
H
2200
F
F
(S)
2500
570
F
F
(R)
20
21
19 diastereomer 1
19 diastereomer 2
2.9
0.64
7800
3800
1500
720
19
15
Compounds ( )-15–19, 20 and 21 were >10 lM for the S1P2 receptor.
(a marker for immunosuppressive efficacy) was deter-
mined in mice, at 3 h post oral administration. Quantifi-
cation of circulating PBLs in this manner also provides a
gauge on the overall pharmacokinetic properties of new
compounds.13
The a-amino acids ( )-9a and ( )-9b were found to be
moderately potent S1P1 agonists. Homologation to the
b-amino acids ( )-11a and ( )-11b led to an improve-
ment in SlP1 potency, with the cis-isomer ( )-11a being
roughly 2-fold more potent than the trans-isomer

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V. J. Colandrea et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2905–2908
Table 3. Rat pharmacokineticsa for selected compounds
compounds have also been shown to lower circulating
PBLs after oral administration in mice and display
encouraging pharmacokinetic profiles in rats.
Compound
Rat pharmacokinetic parameters
11a
Clp = 6.6 mL/min/kg, Vd = 2.9 L/kg,
t1/2 = 4.1 h; % F = 48
11b
21
Clp = 8.4 mL/min/kg, Vd = 3.0 L/kg,
t1/2 = 3.5 h; % F = 40
References and notes
Clp = 9.7 mL/min/kg, Vd = 2.3 L/kg,
t1/2 = 2.2 h; % F = 48
1. Mandala, S.; Hajdu, R.; Bergstrom, J.; Quackenbush, E.;
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a Sprague–Dawley rats (n = 2); 1.0 mg/kg iv; 2.0 mpk po.
( )-11b. Additionally, the cis-isomer ( )-11a was more
selective for the remaining S1P subtypes (>3000-fold
over S1P3, >300-fold over S1P4, and >60-fold over
S1P5); such trends had been observed for analogs of 25
and with 2,4-(disubstituted)pyrrolidine S1P receptor
agonists,14 but were somewhat less pronounced. The c-
amino acids ( )-14a and ( )-14b were approximately
2-fold less potent than b-amino acids ( )-11a and ( )-
11b with excellent selectivity against S1P3 and S1P4,
although both compounds had an increased affinity for
the S1P5 receptor.
2. Matloubian, 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.
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J.; Milligan, J. A.; Shei, G.-J.; Chrebet, G.; Bergstrom, J.;
Card, D.; Rosen, H.; Mandala, S. M. Bioorg. Med. Chem.
Lett. 2004, 14, 4861.
5. Li, Z.; Chen, W.; Hale, J. J.; Lynch, C. L.; Mills, S. G.;
Hajdu, R.; Keohane, C. A.; Rosenbach, M. J.; Milligan, J.
A.; Shei, G.-J.; Chrebet, G.; Parent, S. A.; Bergstrom, J.;
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Med. Chem. 2005, 48, 6169.
6. Hale, J. J.; Doherty, G.; Toth, L.; Mills, S. G.; Hajdu, R.;
Keohane, C. A.; Rosenbach, M. J.; Milligan, J. A.; Shei,
G.-J.; Chrebet, G.; Bergstrom, J.; Card, D.; Forrest, M.;
Sun, S.-Y.; West, S.; Xie, H.; Nomura, N.; Rosen, H.;
Mandala, S. M. Bioorg. Med. Chem. Lett. 2004, 74, 3501.
7. Coudert, E.; Acher, F.; Azerad, R. Synthesis 1997, 863.
8. (a) Ezquerra, J.; Pedregal, C.; Rubio, A.; Valenciano, J.;
Navio, J. L. C.; Builla, J. A.; Vaquero, J. J. Tetrahedron
Lett. 1993, 34, 6317; (b) Ohta, T.; Hosoi, A.; Kimura, T.;
Nozoe, S. Chem. Lett. 1987, 2091.
Administration of either pyrrolidine ( )-11a or ( )-11b
to mice at 10 mpk resulted in a near maximal lowering
of circulating PBLs and both of these compounds exhib-
ited favorable pharmacokinetics in the rat (Table 3).
Based on the in vitro potency and selectivity of the cis-
isomer ( )-11a, additional analogs were prepared in this
series. Chemistry was carried out as outlined in Scheme
3, starting with the amidoxime derived from nitrile ( )-
10a and some select substituted benzoic acids.15 S1P
receptor data for these compounds are listed in Table 2.
4-(Cycloalkyl)phenyl substituents appended to the 5-po-
sition of the 1,2,4-oxadiazole ring had been previously
demonstrated to either maintain or enhance S1P recep-
tor affinity as compared to the corresponding 4-(2-meth-
ylpropyl)phenyl analogs in other series of S1P receptor
agonists. The same trend was observed for the com-
pounds in this study (Table 2). Cyclohexyl analog ( )-
15 and the diastereomeric mixtures 18 and 19 were all
found to have S1P1 IC50 values of 1 nM or less. Since
the (R)-(2,2-difluoro)cyclopentyl analog 19 appeared to
be more potent in the mouse peripheral lymphocyte low-
ering (PLL) screening assay; separation of this com-
pound by chiral HPLC16 afforded the single
diastereomers 20 and 21. Analog 21 was found to be a
potent S1P1 agonist (S1P1 IC50 = 0.6 nM) with greater
than 5900-fold selectivity over S1P3 and 1100-fold selec-
tivity over the S1P4 receptor. Compound 21 displayed
good rat pharmacokinetic properties (Table 3) and its
PLL pharmacodynamic ED50 value in the mouse was
approximately 0.3 mg/kg after oral administration.
9. Van Betsbrugge, J.; Van Den Nest, W.; Verheyden, P.;
´
Tourwe, D. Tetrahedron 1998, 54, 1753.
10. Podlech, J.; Seebach, D. Liebigs Ann. Chem. 1995, 7, 1217.
11. Brown, H. C.; Narasimham, S.; Choi, Y. M. J. Org. Chem.
1982, 47, 4702.
12. Mancuso, A. J.; Swern, D. Synthesis 1981, 165.
13. 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. M. Bioorg. Med. Chem. Lett. 2004, 14,
3351.
14. Yan, L.; Budhu, R.; Huo, P.; Lynch, C. L.; Hale, J. J.;
Mills, S. G.; Hajdu, R.; Keohane, C. A.; Rosenbach, M.
J.; Milligan, J. A.; Shei, G.-J.; Chrebet, G.; Bergstrom, J.;
Card, D.; Mandala, S. M. Bioorg. Med. Chem. Lett.,
accepted for publication.
15. (4-Cyclohexyl)phenylbenzoic acid was purchased from
Lancaster synthesis. For the preparation of benzoic acids
used in the synthesis of compounds 16–19, see Ref. 4.
16. Chiral HPLC was performed on the N-tert-butoxycar-
bonylmethyl ester of compound 19 (Chiralpak OD
column, 2 · 25 cm, 10% EtOH/heptane, 8.0 mL/min,
k = 254 nm, peak 1 = 21.9 min; peak 2 = 26.1 min).
Sequential deprotection afforded compounds 20 and
21.
In short, we have demonstrated that a rational combina-
tion of compounds ( )-1 and 2 led to the identification
of a series of cis-2,5-disubstituted pyrrolidine carboxy-
lates as agonists of the S1P1 receptor, with good to
exceptional selectivity over S1P2,3,4 subtypes. Select