Exploring amino acids derivatives as potent, selective, and direct agonists of sphingosine-1-phosphate receptor subtype-1

Article abstract of DOI:10.1016/j.bmcl.2012.11.053

In the quest to discover a potent and selective class of direct agonists to the sphingosine-1-phosphate receptor, we explored the carboxylate functional group as a replacement to previously reported lead phosphates. This has led to the discovery of potent and selective direct agonists with moderate to substantial in vivo lymphopenia. The previously reported selectivity enhancing moiety (SEM) and selectivity enhancing orientation (SEO) in the phenylamide and phenylimidazole scaffolds were crucial to obtaining selectivity for S1P receptor subtype 1 over 3.

Full text of DOI:10.1016/j.bmcl.2012.11.053

Bioorganic & Medicinal Chemistry Letters 23 (2013) 472–475
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Exploring amino acids derivatives as potent, selective, and direct agonists
of sphingosine-1-phosphate receptor subtype-1
Ghotas Evindar a, , Hongfeng Deng a, Sylvie G. Bernier b, Elisabeth Doyle b, Jeanine Lorusso b,
⇑
Barry A. Morgan a, William F. Westlin b
a Department of Medicinal Chemistry, Praecis Pharmaceuticals Incorporated (Currently GlaxoSmithKline), 830 Winter Street, Waltham, MA 02451, United States
b Department of Preclinical Research, Praecis Pharmaceuticals Incorporated (Currently GlaxoSmithKline), 830 Winter Street, Waltham, MA 02451, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
In the quest to discover a potent and selective class of direct agonists to the sphingosine-1-phosphate
receptor, we explored the carboxylate functional group as a replacement to previously reported lead
phosphates. This has led to the discovery of potent and selective direct agonists with moderate to sub-
stantial in vivo lymphopenia. The previously reported selectivity enhancing moiety (SEM) and selectivity
enhancing orientation (SEO) in the phenylamide and phenylimidazole scaffolds were crucial to obtaining
selectivity for S1P receptor subtype 1 over 3.
Received 29 September 2012
Revised 11 November 2012
Accepted 14 November 2012
Available online 27 November 2012
Keywords:
Sphingosine-1-phosphate (S1P) agonist
S1P1 agonists
Ó 2012 Elsevier Ltd. All rights reserved.
Amino acids derivatives
Phenylamide
Phenylimidazole
Immunosuppressants
Lymphopenia
Exploration of sphingosine-1-phosphate (S1P) receptors, a class
of G-protein coupled receptors (GPCRs), as a potential therapeutic
target in various physiological and pathophysiological processes1
has generated a highly competitive area of research and develop-
ment. S1P, the endogenous natural ligand, effects activation and
inhibition of this cluster of five receptors (S1P1–5) inducing multi-
ple effects on cardiovascular and immune system function.2
FTY-720 (Fingolimod), a novel molecular entity, approved under
the trade name of Gilenya for treatment of relapsing-remitting
multiple sclerosis,3 has allowed for better understanding of this
class of GPCR.4 FTY-720 needs to be phosphorylated to the desired
phosphate drug, FTY-720-phosphate, to interact with S1P receptor
subtypes. Recently, we published several novel classes of S1P
receptor subtype-1 agonists that fall within the same class of
pro-drug approach as FTY-720 that require in vivo phosphorylation
to influence S1P receptors.5 One major challenge in a pro-drug
strategy in S1P activation is in vivo phosphorylation of the pro-
drug to the desired phospho-drug. The challenges of low phosphor-
ylation and variability observed in the in vivo phosphorylation of
the lead molecules in the pro-drug approach led to consideration
of exploring the potential for a direct agonist approach. The use
of a carboxylate as direct S1P receptor subtype-1 agonist was re-
H2N
Me
Me
H
N
OH
OH
PPI-4621
PPI-4667
O
O
H2N
H
N
O
O
F3C
H2N
O
Me
OH
N
PPI-4955
NH
Scheme 1.
cently reported by Mandala and co-workers.6 Utility of a carboxyl-
ate as a potential phosphate mimic led to reinvestigation of the
previously reported pro-drug chemotypes and reconsideration of
the corresponding carboxylate analogs as potential novel thera-
peutic agents. In an earlier report, detailed biological data on lead
alcohol PPI-4621 (Scheme 1) was disclosed.5 The low in vivo con-
version of the pro-drug PPI-4621 to the corresponding potent
phosphate (Table 1) and poor selectivity of PPI-4621-phosphate
for S1P subtype-1 over 3 were the drawback to further progress
of this promising lead. Thus, further SAR exploration of the lipo-
philic tail moiety of PPI-4621-phosphate led to discovery of potent
⇑
Corresponding author.
E-mail address: ghotas.x.evindar@gsk.com (G. Evindar).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.053

G. Evindar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 472–475
473
and selective PPI-4667-phophate with a better selectivity profile
for S1P subtype-1 over 3. However, the drawback to PPI-4667 alco-
hol came with its poor in vivo conversion to the corresponding
phosphate in a rat model. Further structure–activity relationship
studies resulted in discovery of PPI-4955 with efficient rodent
in vivo phosphorylation and excellent potency and selectivity pro-
files. Unfortunately, although PPI-4955 was shown to have a high
degree of phosphorylation in vivo in rodents, it demonstrated poor
in vivo phosphorylation in higher species. Therefore, two routes
were considered to overcome the phosphorylation predicament;
(a) direct agonist approach, (b) extensive exploration in the pro-
drug approach to further improve in vivo phosphorylation of the
desired alcohol to phosphate in higher species. In this Letter, we
describe the direct agonist approach.
Table 1
Percent in vivo pro-drug coversion to the corresponding phosphate upon 10 mg/kg
oral (PO) administration of the pro-drug alcohol
Pro-drug
% Phosphate in mice
% Phosphate in rats
PPI-4621
PPI-4667
PPI-4955
10
70
60
20
14
49
In pursuit of a carboxylate as a direct S1P receptor subtype-1
agonist, we initiated synthesis of the carboxylate corresponding to
the previously reported phosphate lead molecules as shown in
Scheme 2.7 Acylation of the amino group of the substituted aniline
1 with the desired orthogonally protected amino acid using
O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexa-
fluorophosphate (HATU) and N,N-diisopropylethylamine (DIPEA)
afforded esters 2 and 4 (Scheme 2a).8 The corresponding des-oxy
analog of 1 was synthesized from Sonogashira cross-coupling of aryl
iodide 5 with 1-octyne followed by hydrogenation of both the nitro
and the alkyne in one step to afford compound 6. Coupling of the
aniline 6 to the desired orthogonally protected amino acids was
achieved analogous to aniline 1 to afford compound 2. Two step
deprotection of the intermediates 2 and 4 gave the desired final
compounds 3 and 3f, respectively. Additional carboxylate analogs
were synthesized as described in Scheme 2b. Swern oxidation of
alcohol 7 provided aldehyde 8 which upon a Horner–Wadsworth–
Emmons reaction afforded olefin 9. Hydrogenation of the olefin
residue produced compound 10. Two step deprotection of the
functional groups in 9 and 10 gave acids 3d and 3c, respectively.
The synthesized acids where then evaluated for S1P receptor
binding activity and in vivo activity (Table 2). The binding activity
of the designed agonists was measured using a [33P] binding assay
as described in an earlier report.5a The carboxylate corresponding
to the lead molecule PPI-4621 (3a) demonstrated excellent binding
activity at S1P receptor subtype-1 and 73-fold selectivity for recep-
tor subtype-1 over 3. Compound 3a also provided potent in vivo
activity with a 57% decrease in circulating lymphocytes following
an oral dose of 10 mg/kg. Changing the amino acid aspartate (3a)
to glutamate (3b) decreased S1P receptor subtype-1 binding activ-
ity by sixfold and decreased in vivo lymphopenia activity. Modifi-
NO2
I
CF3
(a)
5
v, ii
NH2
NH2
Cbz
C8H17
O
C8H17
R1
1
CF3
6
i
i
iv
Boc
NH
R2
NH
Me
O
H
O
H
N
N
X
OBn
OtBu
O
C8H17
O
C8H17
Y
O
R1
CF3
2
4
ii, iii
ii, iii
H2N
H2N
O
O
R2
X
H
N
H
Me
N
OH
OH
O
O
C8H17
C8H17
O
Y
R1
CF3
3
3f
(b)
Boc
Boc
NH
Me
NH
Me
cation of the glutamic acid 3b to
a-methyl-glutamate (3c)
H
ii
H
i
N
N
OH
O
maintained similar receptor potency and selectivity, however,
these compounds lacked any an effect on circulating lymphocytes
upon oral administration. The corresponding olefin 3d also gave
analogous binding activity and lymphopenia.
C8H17
O
C8H17
O
O
O
8
7
Boc
Boc
NH
Me
NH
Table 2
[
H
H
33P] Binding activity on S1P1 and S1P3 receptor subtypes and percent lymphopenia
iii
Me
N
N
O
O
Me
Me
obtained upon 10 mg/kg oral (PO) administration of the agonist at 6 h post-dosing in
C8H17
O
mice10
C8H17
O
O
O
O
O
9
10
H2N
O
R2
X
H
N
iv, v
iv, v
OH
C8H17
O
Y
R1
H2N
H2N
H
N
Me
H
N
R1
R2
Y
X
hS1P1 IC50 (nM) hS1P3 IC50 (nM) %La
Me
A
OH
OH
C8H17
O
3a
3b
3c
3d
H
H
H
H
H
H
Me
Me
H
O
O
O
O
O
O
—
CH2
14.4
92
77
74
53
1050
57
44
N
25
33
24
—
C8H17
O
O
O
O
O
CH2CH2
CH2CH2
CH@CH (E)
CH2
CH2
CH2
10,000
10,000
10,000
>10,000
>10,000
>10,000
3d
3c
Scheme 2. Reagents and conditions: (a) (i) N-Boc-amino acid benzyl ester, HATU,
DIPEA, DMF; (ii) 10% Pd on carbon, H2 (gas), MeOH; (iii) TFA, CH2Cl2; (iv) (S)-2-
(benzyloxycarbonylamino)-4-tert-butoxy-2-methyl-4-oxobutanoic acid,9 HATU,
DIPEA, DMF; (v) 1-octyne, Pd(dba)2, PPh3, CuI, DIEA, MeCN. (b) (i) DMSO, oxalyl
chloride, Et3N, CH2Cl2; (ii) (methoxycarbonylmethyl)triphenyl-phosphonium chlo-
ride; (iii) 10% Pd on carbon, H2 (gas), MeOH; (iv) LiOH, MeOH, H2O, THF; (v) TFA,
CH2Cl2.
3e CF3
3f
3g CF3 Me
CF3 Me
95
370
a
%L = Lymphopenia (percent decrease in circulating lymphocytes compared to
time-matched vehicle control).
bN = negligible.

474
G. Evindar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 472–475
In order to improve binding activity and in vivo lymphopenia
profile of the agonist while maintaining S1P receptor subtype
selectivity, we utilized our previous learning of the pro-drug ap-
proach and decided to modify the amide component to a more ri-
gid imidazole in our exploration. Therefore the corresponding
imidazole analogs of PPI-4955 were synthesized as described in
Scheme 3. Reaction of the bromoacetophenone 11 with orthogo-
nally protected amino acid or mono-protected dicarboxylate pro-
vided the ester intermediate which upon intramolecular
cyclization in the presence of excess ammonium acetate, afforded
the desired phenylimidazole 12. Removal of the protecting
group(s) in phenylimidazole 12 afforded the desired final product
13.
CF3
CF3
O
O
i, ii
R1
N
R2
Br
O
O
NH
O R3
11
12
CF3
O
R1
iii or iv
N
R2
O
NH
OH
13
The in vitro and in vivo evaluation of the synthesized phenyl-
imidazole carboxylates is reported in Table 3. The corresponding
phenylimidazole analog of PPI-4955 (13a) showed an S1P receptor
subtype-1 binding activity of 82 nM with no detectable activity at
S1P receptor subtype-3. However, compound 13a also gave no
detectable lymphopenia upon 10 mg/kg oral administration.
Changing R2 from a methyl to an H improved the S1P receptor sub-
type-1 binding activity to 17 nM, with no binding activity at S1P
receptor subtype-3, but no lymphopenia was observed upon oral
administration of 13b either. Further modification of the amino
acid portion of the agonist by replacement of the amino group
(R1 = NH2) with an H, diminished the agonist activity, indicating
that the imidazole NH could not replace the required interaction
observed with the primary amine required to obtain the potent
agonist activity. The overall observation of lack of lymphopenia
with the imidazole carboxylate was suggestive of the need for fur-
ther SAR exploration of the amide carboxylate series.
Scheme 3. Reagents and conditions: (i) butanoic acid or protected 2-amino-
butanoic acid as mono-ester, Cs2CO3, DMF; (ii) AcONH4, toluene, 100 °C (iii) 10% Pd
on carbon, H2 (gas), MeOH (where R1 = NHCbz or R3 = Bn); (iv) TFA, CH2Cl2 (where
R1 = NHBoc or R3 = tBu).
Table 3
[
33P] Binding activity on S1P1 and S1P3 receptor subtypes and percent lymphopenia
obtained upon 10 mg/kg oral (PO) administration of the agonist at 6 h post-dosing in
mice10
CF3
O
R1
N
R2
O
NH
OH
In order to reconfirm the agonist potency and selectivity, a
number of designed carboxylates were further investigated in
Carboxylate
R1
R2
hS1P1 IC50 (nM)
hS1P3 IC50 (nM)
%La
13a
13b
13c
NH2
NH2
H
Me
H
H
82
17
3000
>10,000
>10,000
>10,000
N
N
N
[c-
35S]GTP functional assays as reported in Table 4.11 The amide
3a, analogous to binding activity data, showed a relatively lower
in vitro activity and selectivity for S1P subtype-1 over 3. However,
insertion of the SEM group, with required SEO, in 3e and 3f dimin-
ished S1P receptor subtype-3 activity, providing agonist selectivity
while maintaining potent activity at receptor subtype-1. The corre-
sponding imidazole to PPI-4955 (13a) showed fivefold weaker
activity at S1P receptor subtype-1 in comparison to any of the
amide analogs. Replacement of the R2 methyl with a H, improved
the S1P receptor subtype 1 activity in the imidazole scaffold similar
to the amide scaffold level. However, overall, the data in the func-
tional assay and the lymphopenia indicated the necessity for fur-
ther improvement in direct agonist activity at S1P receptor
subtype-1.
a
%L = Lymphopenia (percent decrease in circulating lymphocytes compared to
time-matched vehicle control).
bN = negligible.
Incorporation of the selectivity enhancing moiety (SEM), with
previously reported selectivity enhancing orientation (SEO), in 3a
gave compound 3e with improved agonist selectivity. Compound
3e provided moderate lymphopenia (60%) upon 20 mg/kg intrave-
nous (iv) administration at 6 h post-dosing in mice, indicating the
necessity for further improvement in agonist potency at S1P recep-
tor subtype-1. Use of an unusual
a-methyl amino acid in 3e pro-
In summary, in an attempt to develop the corresponding car-
boxylates to our earlier reported pro-drug lead molecules and to
overcome the in vivo phosphorylation issue in the pro-drug strat-
egy, we discovered a few carboxylates with excellent activity at
vided 3f, which demonstrated similar binding activity, receptor
selectivity, and lymphopenia profile as 3e. However, the des-oxy
analog (3g) demonstrated poor binding activity, indicating the
importance of the oxygen group in the tail portion of the agonist.
Table 4
[c-
35S]GTP functional activity on S1P1 and S1P3 receptor subtypes
R2
R1
O
H2N
O
R3
Y
OH
Agonist
R1
R2
R3
Y
hS1P1 EC50 (nM)
hS1P3 EC50 (nM)
S1P3/S1P1
S1P
3a
3e
3f
13a
13b
—
—
H
CF3
CF3
CF3
CF3
—
H
H
Me
Me
H
—
Amide
Amide
Amide
Imidazole
Imidazole
5.6
2.4
—
11
>13
>22
>4
C7H13
C7H13
C7H13
164
231
135
700
161
1800
>3000
>3000
>3000
>3000
>18.6

G. Evindar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 472–475
475
Montalban, X.; O’Connor, P.; Polman, C. H.; Haas, T.; Korn, A. A.; Karlsson, G.;
Radue, E. W. N. Eng. J. Med. 2006, 355, 1124; (c) Khoury, S. J. Watch Neurol. 2006,
1, 1.
S1P receptor subtype-1. The concept of the SEM group with appro-
priate SEO that was established in an earlier report5c was once
again demonstrated to improve agonist selectivity for receptor
subtype-1 over 3. The amide direct agonists demonstrated moder-
ate to substantial lymphopenia upon oral administration. However,
further lead optimization is necessary to improve the binding
activity and in vivo activity (lymphopenia) of the current carboxyl-
ate lead molecules.
5. (a) Evindar, G.; Bernier, S. G.; Kavarana, M. J.; Doyle, E.; Lorusso, J.; Kelley, M. S.;
Halley, K.; Hutchings, A.; Wright, A. D.; Saha, A. K.; Hannig, G.; Morgan, B. A.;
Westlin, W. F. Bioorg. Med. Chem. Lett. 2009, 19, 369; (b) Evindar, G.; Satz, A. L.;
Bernier, S. G.; Kavarana, M. J.; Doyle, E.; Lorusso, J.; Taghizadeh, N.; Halley, K.;
Hutchings, A.; Kelley, M. S.; Wright, A. D.; Saha, A. K.; Hannig, G.; Morgan, B. A.;
Westlin, W. F. Bioorg. Med. Chem. Lett. 2009, 19, 2315; (c) Evindar, G.; Bernier, S.
G.; Doyle, E.; Kavarana, M. J.; Satz, A. L.; Lorusso, J.; Blanchette, H. S.; Saha, A. K.;
Hannig, G.; Morgan, B. A.; Westlin, W. F. Bioorg. Med. Chem. Lett. 2010, 20, 2520.
6. 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.
7. For synthesis of compounds 1 and 5 refer to Ref. 4a.
8. For synthesis of aniline 1 refer to Ref. 4a.
9. (S)-2-(Benzyloxycarbonylamino)-4-tert-butoxy-2-methyl-4-oxobutanoic acid
was synthesized according to: Altmann, E.; Nebel, K.; Mutter, M. Helv. Chim.
Acta 1991, 74, 800.
References and notes
1. Young, N.; Van Brocklyn, J. R. Sci. World J. 2006, 6, 946.
2. (a) Spiegel, S.; Milstein, M. Nat. Rev. Mol. Cell Biol. 2003, 4, 397; (b) Spiegel, S.;
Milstein, S. Biochem. Soc. Trans. 2003, 31, 1216.
3. (a) http://www.drugs.com/gilenya.html.; (b) FDA press release on approval of
Gilenya: http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/
ucm226755.htm.
4. (a) Salvadori, M.; Budde, K.; Charpentier, B.; Klempnauer, J.; Nashan, B.;
Pallardo, L. M.; Eris, J.; Schena, F. P.; Eisenberger, U.; Rostaing, L.; Hmissi, A.;
Aradhye, S. Am. J. Transplant. 2006, 6, 2912; (b) Kappos, L.; Antel, J.; Comi, G.;
10. FTY-720 was used as the control in these experiments. Upon 1.0 mg/kg oral
(PO) administration of FTY-720 generally 80% lymphopenia was observed.
11. For [c-
35S]GTP functional assay protocol refer to Ref. 5.