Synthesis of Para-alkyl aryl amide analogues of sphingosine-1-phosphate: Discovery of potent S1P receptor agonists

Article abstract of DOI:10.1016/S0960-894X(03)00812-6

Sphingosine-1-phosphate (S1P) is a biologically active lysophospholipid with the capacity to induce a broad range of cellular responses via its interaction with the S1P family of G-protein coupled receptors. This report describes the synthesis of several

Full text of DOI:10.1016/S0960-894X(03)00812-6

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3401–3404
Synthesis of Para-Alkyl Aryl Amide Analogues of Sphingosine-1-
phosphate: Discovery of Potent S1P Receptor Agonists
Jeremy J. Clemens,a Michael D. Davis,b Kevin R. Lynchb,c and Timothy L. Macdonalda,*
aDepartment of Chemistry, University of Virginia, McCormick Road, PO Box 400319, Charlottesville, VA 22904, USA
bDepartment of Biochemistry and Molecular Biology, University of Virginia, McCormick Road, PO Box 400319, Charlottesville,
VA 22904, USA
cDepartment of Pharmacology, University of Virginia, McCormick Road, PO Box 400319, Charlottesville, VA 22904, USA
Received 29 April 2003; revised 22 July 2003; accepted 29 July 2003
Abstract—Sphingosine-1-phosphate (S1P) is a biologically active lysophospholipid with the capacity to induce a broad range of
cellular responses via its interaction with the S1P family of G-protein coupled receptors. This report describes the synthesis of
several potent S1P receptor agonists. For instance, compound 9c displayed an EC50=8.6 nM at the S1P1 receptor using a
[g-35S]GTP binding assay as compared to an EC50=4.5 nM for the endogenous ligand. We also report the effects associated with
introduction of a phenyl ring between the ‘linker’ and ‘lipophilic tail’ regions of the analogues, for example total loss of activity at
S1P2 and increased agonism at S1P5.
# 2003 Elsevier Ltd. All rights reserved.
Sphingosine-1-phosphate (S1P) has been demonstrated
to act as both an intercellular signaling molecule and an
intracellular second messenger (Fig. 1).1 Extracellular
S1P binds a specific subset of seven trans-membrane
G-protein coupled receptors, namely the S1P1 (formerly
Edg-1), S1P2 (Edg-5), S1P3 (Edg-3), S1P4 (Edg-6), and
S1P5 (Edg-8) receptors.2 Through its interaction with
these receptors, S1P has been shown to induce a wide
variety of cellular effects including differentiation,
motility, and escape from apoptosis.3
To understand better the processes associated with S1P
binding to its receptors, it is vital to establish a struc-
ture–activity relationship (SAR) for S1P with respect to
each individual receptor. The generation of S1P ana-
logues with different binding profiles across the S1P
receptors proves useful in elucidating this SAR. More
specifically, the synthesis of S1P receptor specific agon-
ists and antagonists could prove useful in the discovery
of novel drugs. To supplement our previously reported
S1P1/S1P3 receptor agonist,7 we now wish to report the
synthesis of potent S1P1, S1P3, S1P4 and S1P5 receptor
agonists along with their activity profiles across all five
recombinant human S1P receptors. We also report sev-
eral interesting effects in activity with the incorporation
The ability of S1P to mediate biological processes such
as immune modulation make S1P receptor agonists and
antagonists attractive candidates for use as therapeutic
agents.4 For example, the sphingosine analogue,
FTY720, is a novel immunosuppressive agent that acts
by reversibly sequestering lymphocytes to secondary
lymphoid tissues.5 FTY720is a pro-drug that, after
O-phosphorylation by a yet to be defined kinase, elicits
lymphopenia via its interaction with one or more of the
S1P receptors.5,6 Phosphorylated FTY720(phospho-
FTY720) shares several structural similarities with S1P
including a lipophilic tail region, a 2-amino group, and
a phosphate head group.
*Corresponding author. Tel. +1-434-924-7718; fax:+434-982-2302;
e-mail: tlm@virginia.edu
Figure 1. Structures and regions of S1P and phospho-FTY720.
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00812-6

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J. J. Clemens et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3401–3404
of a phenyl ring between the ‘linker’ and ‘lipophilic tail’
regions of the S1P analogues, as is the case in phos-
pho-FTY720. These effects include a total loss of
activity at S1P2, increase in agonism at S1P5, and a loss
of C2-stereospecificity at the S1P1 and S1P3 receptors.
commenced with PyBOP mediated coupling of N-Boc-
(d)-Serine(Bzl)–OH with the appropriate alkyl amine
or p-alkyl aniline (Scheme 2).7 The resulting amides
6a–e were then subjected to hydrogenolysis to afford the
deprotected alcohols 7a–e. These alcohols were then
phosphorylated using di-tert-butyl diisopropylpho-
sphoramidite with subsequent oxidation of the phos-
phite by hydrogen peroxide to give the protected
phosphates 8a–e. Deprotection of 8a–e using 1:1 TFA/
CH2Cl2 gave the final products 5e and 9a–d as the TFA
salts.
Our preparation of S1P analogues began with the
synthesis of the (2S)-N-alkyl amide, (2S)-O-alkyl ester,
and (2S)-N-aryl amide compounds, 5a–c, 5d, and 9e–f,
respectively (Scheme 1).7 N-Boc-(l)-Serine was pro-
tected as the benzyl ester using benzyl bromide under
standard conditions. The protected amino acid 1 was
then phosphorylated using di-tert-butyl diisopropyl-
phosphoramidite with subsequent hydrogen peroxide
oxidation of the phosphite to afford 2 which underwent
hydrogenolysis to give acid 3. Coupling of 3 to the
appropriate alkyl amine using DCC afforded the pro-
tected N-alkyl amides 4a–c. Coupling of 3 to 1-tetra-
decanol using the PyBOP reagent gave the protected O-
alkyl ester 4d. Coupling of 3 to the appropriate p-alkyl
aniline using the PyBOP reagent afforded the protected
N-aryl amides 4e–f. Deprotection of the amides 4a–c
and 4e–f and ester 4d using 1:1 TFA/CH2Cl2 gave the
final products 5a–d and 9e–f as the TFA salts.
Receptor activation by S1P and the synthetic analogues
was determined in vitro by measuring the ligand depen-
dant binding of [g-35S]GTP to membranes containing
each of the five human S1P receptors expressed in
HEK293T cells.8 None of the compounds in these series
showed any activity at the related lysophosphatidic acid
receptors (LPA1À3) at concentrations up to 10 mM in
this assay (data not shown). As was previously reported,
R-configuration at the C2 position of the N-alkyl amide
analogues appears to result in increased binding at S1P1
and S1P3, but not at the other S1P receptors (Table 1).7
Although the N-alkyl amide and O-alkyl ester com-
pounds were poor agonists at S1P2, introduction of a
phenyl ring between the ‘linker’ and ‘lipophilic tail’
The synthetic route to the (2R)-N-alkyl amide and (2R)-
N-aryl amide compounds, 5e and 9a–d, respectively,
Scheme 1. Reagents and conditions: (i) Cs2CO3, BnBr, DMF, rt, 12 h, quant.; (ii) tetrazole, di-tert-butyl diisopropylphosphoramidite, 1:1 CH2Cl2/
THF, rt, 12 h; (iii) H2O2, rt, 4 h, quant. (2 steps); (iv) H2, 10% Pd/C, EtOH, rt, 12 h, 91%; (v) DCC, DMAP, HOBT, CH2Cl2, rt, 12 h, 26–41%;
(vi) PyBOP, DIEA, CH2Cl2, rt, 6 h, 43–60% (15% for 4d); (vii) 1:1 TFA/CH2Cl2, rt, 4 h, 96–100%.
Scheme 2. Reagents and conditions: (i) PyBOP, DIEA, CH2Cl2, rt, 6 h, 65–77%; (ii) H2, 10% Pd/C, EtOH, rt, 12 h, 84–96%; (iii) tetrazole, di-tert-
butyl diisopropylphosphoramidite, 1:1 CH2Cl2/THF, rt, 12 h; (iv) H2O2, rt, 4 h, 60–100% (2 steps, 9% for 9d); (v) 1:1 TFA/CH2Cl2, rt, 4 h, 56–
100%.

J. J. Clemens et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3401–3404
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Figure 2. [g-35S]GTP binding to HEK293T cell membranes in response to S1P and N-aryl amide S1P analogues. Each data point represents the
mean of three determinations.
regions of the analogues, as in compounds 9a–f, resulted
in a complete loss of activity at that receptor type (Table
1, Fig. 2). This structural modification also resulted in
increased binding affinity towards S1P5. In contrast to
the N-alkyl amide compounds, stereochemical con-
figuration at the C2 position of the N-aryl amide ana-
logues does not appear to have an effect on activity at
any of the S1P receptors. This finding also differs from
data reported by Lynch, et al. for similar compounds
containing 2-methyl groups, (S)-AFD and (R)-AFD, in
which only the 2R enantiomer acted as a potent agonist
at the S1P receptors (Fig. 3).6
Regarding the S1P1 and S1P3 receptors, the length of
the alkyl chain of the N-aryl amide compounds was
optimized at 10carbons, compound
9c displayed
EC50s=8.6 nM and 89 nM, respectively, on those
receptors. With respect to the S1P4 receptor, shorter
chain lengths of six and eight carbons on the N-aryl
amide compounds exhibited higher potency and efficacy
than the longer chain lengths of 10and 12 carbons. At
the S1P5 receptor, the greatest efficacy of the N-aryl
amide compounds was achieved with the longest chain
length, 12 carbons. The potency, however, of the N-aryl
amide compounds on S1P5 was approximately equal,
about one log order less than that of S1P itself.
To summarize, we have synthesized a second generation
of S1P analogues that incorporate a phenyl ring
between the ‘linker’ and ‘lipophilic tail’ regions of the
pharmacophore, as is the case in phospho-FTY720.
This structural modification has resulted in the genera-
tion of highly potent, fully efficacious agonists on four
of the S1P receptors; S1P1, S1P3, S1P4 and S1P5. In
particular, introduction of the phenyl ring between the
‘linker’ and ‘lipophilic tail’ regions of the S1P analo-
gues described here results in complete loss of activity
at S1P2, increased activity at S1P5, and a loss of C2-
stereospecificity at the S1P1 and S1P3 receptors. These
findings have helped to decipher further the SAR of S1P
on the five S1P receptors and will serve as the basis for
additional studies along this route.
Table 1. EC50 Values (nM) for S1P and N/O-alkyl amide/ester and
N-aryl amide analogues at S1P receptors determined by a [g-35S]GTP
binding assaya,b
N/O-Alkyl Compd
S1P1
S1P2
S1P3
S1P4
S1P5
43.9
S1P
5a
5b
5c
5d
5e
0.9
598.4
397.02685.0862.4
1805.0 >5000
322.1
12.7
2.9
973.2
1.1
845.4
nd 6.0160
878.6
601.9
50.8
nd
nd
645.5
nd
1220.0
2760.0
2107.0
nd >5000
nd >5000
N-Aryl Compd
S1P1
S1P2
S1P3
S1P4
S1P5
S1P
9a
9b
9c
9d
9e
4.5
260na
58
8.3
na
na
na
174400
n5a05
na
8.7
88403
04505520
89
2709.2
45041
910
8.6
130n1a600
550na
82
150
1500
Acknowledgements
9f
110
aValues are means of three experiments (na=not active, nd=no data).
bData for N/O-alkyl compounds are taken from ref 7.
This work was supported by grants from the NIH
[NIGMS R01 GM052722 (to K.R.L.) and NIGMS F31
GM064101 (to M.D.D.)].
References and Notes
1. For a short review on S1P as a signaling molecule, see:
Speigel, S.; Milstien, S. J. Biol. Chem. 2002, 277, 25851.
2. Chun, J.; Goetzl, E. J.; Hla, T.; Igarashi, Y.; Lynch, K. R.;
Moolenaar, W.; Pyne, S.; Tigyi, G. Pharmacol. Rev. 2002, 54, 265.
3. (a) Goetzl, E. J.; Lee, H.; Dolezalova, H.; Kalli, K. R.;
Conover, C. A.; Hu, Y. L.; Azuma, T.; Stossel, T. P.; Karliner,
J. S.; Jaffe, R. B. Ann. N.Y. Acad. Sci. 2000, 905, 177. (b)
Figure 3. Structures of (S)-AFD and (R)-AFD (see ref 6).

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J. J. Clemens et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3401–3404
Sugimoto, N.; Takuwa, N.; Okamoto, H.; Sakurada, S.;
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7. For information on the N/O-alkyl amide/ester compounds,
including more detailed experimentals, see: Im, D. S.; Clem-
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8. Brief assay protocol: S1P was purchased from Biomol
(Plymouth Meeting, PA). [g-35S]GTP was purchased from
New England Nuclear (Boston, MA). HEK293T cells were
a gift from Dr. Judy White (Department of Cell Biology,
University of Virginia). First, 5 mg of membranes from the
appropriate receptor and G-protein expressing HEK293T cells
were incubated in 0.1 mL of GTP-binding buffer (in mM:
HEPES 50, NaCl 100, MgCl2 5), pH 7.5 containing 5 mg sapo-
nin, 10 mM GDP, 0.1 nM [g-35S]GTP (1200 Ci/mmol) and test
lipid. After incubating for 30min at 30 ꢀC, bound radionuclide
was separated from free by filtration through Whatman GF/C
paper using a Brandel Cell Harvester (Gaithersburg, MD).
Bound radionuclide was detected with a Packard Top Count
liquid scintillation counter.