Synthesis of benzimidazole based analogues of sphingosine-1-phosphate: Discovery of potent, subtype-selective S1P4 receptor agonists

Article abstract of DOI:10.1016/j.bmcl.2004.07.030

We report the synthesis and potencies of several selective S1P₄ receptor agonists. Sphingosine-1-phosphate (S1P) is a biologically active lysophospholipid with the capacity to induce a broad range of cellular responses via its interaction with

Full text of DOI:10.1016/j.bmcl.2004.07.030

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4903–4906
Synthesis of benzimidazole based analogues of
sphingosine-1-phosphate: discovery of potent,
subtype-selective S1P4 receptor agonists
b
b,c
Jeremy J. Clemens,a, Michael D. Davis, Kevin R. Lynch and Timothy L. Macdonalda
*
aDepartment of Chemistry, University of Virginia, Charlottesville, VA 22903, USA
bDepartment of Biochemistry and Molecular Biology, University of Virginia, Charlottesville, VA 22903, USA
cDepartment of Pharmacology, University of Virginia, Charlottesville, VA 22903, USA
Received 10 May 2004; revised 15 July 2004; accepted 17 July 2004
Available online 7 August 2004
Abstract—Sphingosine-1-phosphate (S1P) is a biologically active lysophospholipid with the capacity to induce a broad range of cel-
lular responses via its interaction with the S1P family of G-protein coupled receptors. A member of this receptor family, S1P4, is
highly and almost exclusively expressed in the lymphoid system and has been implicated in regulation of cell shape and motility.
This report describes the synthesis of several potent benzimidazole based S1P4 receptor selective agonists. For instance, compound
9b displayed an EC50 = 36nM at the S1P4 receptor using a [c-35S]GTP binding assay as compared to an EC50 = 37nM for the
endogenous ligand. We also report the effects of altering stereochemistry at the C2 position, methylation at the C1 and C2 position,
and activity differences between the alcohol and phosphate head groups of the analogues.
Ó 2004 Elsevier Ltd. All rights reserved.
Sphingosine-1-phosphate (S1P) acts as both an intercel-
lular mediator and an intracellular second messenger
(Fig. 1).1 Although the molecular target of intracellular
S1P remains to be defined, extracellular S1P binds a spe-
cific subset of seven trans-membrane G-protein coupled
receptors, namely S1P1–5.2 Through its interaction with
these receptors, S1P has been shown to induce a wide
variety of cellular effects including differentiation, motil-
ity, and escape from apoptosis.3 Recently, S1P receptor
activation has been implicated in regulation of immuno-
Figure 1. Structures and regions of S1P and PhS1P.
suppression through studies on the novel immunomod-
ulator FTY720 and several analogues.4 Agonism at
S1P3 has been shown to produce acute toxicity and reg-
The G-protein coupled receptor S1P4, unlike the other
ulate cardiovascular function whereas agonism at S1P1
S1P receptors, has been shown to be predominately
has been implicated in the regulation of lymphocytes
expressed in the lymphoid system.5 Recently S1P4 was
via sequestration from the blood to secondary lymphoid
implicated in regulation of cell shape and motility via
tissues. Nevertheless, understanding of individual S1P
coupling to Gai and Ga12/13 subunits of heterotrimeric
receptors remains limited by the lack of selective agon-
G-proteins.6 To date, no selective agonist for S1P4 has
been reported, although phytosphingosine-1-phosphate
ists and antagonists for these receptors.
(PhS1P), a naturally occurring minor component of
S1P species, has been established as a high affinity agon-
ist at S1P4.7 To supplement our previously reported
S1P receptor agonists,8 we report now the synthesis
Keywords: Sphingosine-1-phosphate; S1P; S1P4; S1P analogues; S1P
receptor agonists; FTY720.
*
of potent, benzimidazole based S1P4 selective agon-
ists along with their activity profiles across all five
Corresponding author. Tel.: +1-310-825-0549; fax: +1-310-206-
4038; e-mail: clemens@chem.ucla.edu
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.07.030

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J. J. Clemens et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4903–4906
recombinant human S1P receptors. We report also the
effects of alternating stereochemistry of the C2 amino
group, differences in agonism between the alcohol and
phosphate head groups, and the effects of methylation
at the C1 and C2 position of the analogues.
pound, which did not undergo cyclization–dehydration
(Scheme 2). Treatment of this crude mixture with
LawessonÕs reagent effectively completed the conversion
to 10. Birch reduction of the benzyl protected alcohol 10
supplied the free alcohol 11, which was converted to the
final benzimidazole based 1-methyl alcohol 12 by treat-
ment with strong acid. The route to the benzimidazole
based 1-methyl phosphate compound continued with
phosphorylation of the secondary alcohol 11 followed
by oxidation employing hydrogen peroxide and ensuing
global deprotection to afford 14 as the TFA salt.
Our preparation of S1P analogues initiated with the syn-
thesis of the benzimidazole based 7a, 7b, 9a, and 9b
compounds (Scheme 1). 4-Octylaniline was protected
as the mono-acetate affording the desired electronic con-
ditions for mono ortho-nitration of the ring to give 2.
The protected ortho-nitro aniline 2 was then deprotected
in strong base and subsequently reduced to the diamine
4, which was used rapidly in ensuing steps after isola-
tion. Coupling of 4 to the stereochemically appropriate
amino acid using the PyBOP reagent afforded, after con-
sequential cyclization, the protected benzimidazole com-
pounds 5a and 5b. To the best of our knowledge, this is
the first example of the PyBOP reagent promoting a
condensation–cyclization–dehydration event to give a
benzimidazole, albeit in unoptimized yield. Hydrogeno-
lysis of 5a and 5b afforded the N-Boc protected alcohols
6a and 6b, respectively. The final alcohol compounds 7a
and 7b were obtained by deprotection of 6a and 6b,
respectively, in strong acid. The route to the phosphate
compounds continued with phosphorylation of the
alcohols 6a and 6b followed by oxidation utilizing
hydrogen peroxide and finally global deprotection in
strong acid to yield 9a and 9b, respectively, as the
TFA salts.
Synthesis of the racemic 2-methyl benzimidazole ana-
logue 18 initiated with Fisher esterification of a-
methyl-(DL)-serine followed by N-Boc protection to give
alcohol 15 (Scheme 3). Phosphorylation of 15 with sub-
sequent oxidation by hydrogen peroxide followed by
saponification of the crude protected phosphate gave
the suitably protected acid 16. Coupling of compound
4 (synthesis described in Scheme 1) to acid 16 using
the PyBOP reagent gave, after resulting cyclization,
the protected benzimidazole compound 17, which was
then wholly deprotected in strong acid to provide the
benzimidazole based 2-methyl phosphate 18 as the
TFA salt.
Receptor activation by S1P and the synthetic analogues
was determined in vitro by measuring the ligand
dependent binding of [c-35S]GTP to membranes con-
taining each of the five human S1P receptors expressed
in HEK293T cells.9 None of the compounds in these ser-
ies showed any activity at the related lysophosphatidic
acid receptors (LPA1–3) at concentrations up to 10lM
in this assay (data not shown). In comparison to our
previously reported N-alkyl amide, O-alkyl ester, and
N-aryl amide S1P analogues, agonistic activity at S1P1,
S1P3, and S1P5 has been drastically reduced with incor-
The synthetic route to the 1-methyl benzimidazole
analogues 12 and 14 commenced with coupling of the
intermediate 4 (synthesis described in Scheme 1) with
N-Boc-Thr(Bzl)-OH using the PyBOP reagent affording
a chromatographically inseparable mixture of cyclized
benzimidazole 10 and the mono-coupled amide com-
Scheme 1. Reagents and conditions: (i) Ac2O, rt, 1h, quant.; (ii) HNO3, HOAc, Ac2O, ꢀ15 ! 0°C, 3h, quant.; (iii) 40% KOH, EtOH, reflux, 1h,
84%; (iv) Zn dust, HOAc, rt, 2h, 92%; (v) PyBOP, DIEA, CH2Cl2, rt, 6h, 17–33%; (vi) H2, 10% Pd/C, cat. HCOOH, EtOH, rt, 12h, 47–57%; (vii) 1:1
TFA/CH2Cl2, rt, 4h, 71–90%; (viii) tetrazole, di-tert-butyl diisopropylphosphoramidite, 1:1 CH2Cl2/THF, rt, 12h; (ix) H2O2, rt, 4h, 32–52% (two
steps); (x) 1:1 TFA/CH2Cl2, rt, 4h, 62–74%.

J. J. Clemens et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4903–4906
4905
Scheme 2. Reagents and conditions: (i) PyBOP, DIEA, CH2Cl2, rt, 6h; (ii) [2,4-bis(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane-2,4-disulfide],
THF, reflux, 5h, 30% (two steps); (iii) Na0, NH3, THF, ꢀ78°C, 1h, 63%; (iv) 1:1 TFA/CH2Cl2, rt, 4h, 86%; (v) tetrazole, di-tert-butyl
diisopropylphosphoramidite, 1:1 CH2Cl2/THF, rt, 12h; (vi) H2O2, rt, 4h, 16% (two steps); (vii) 1:1 TFA/CH2Cl2, rt, 4h, quant.
Scheme 3. Reagents and conditions: (i) SOCl2, MeOH, rt, 6h; (ii) Boc2O, satd aq NaHCO3, p-dioxane, rt, 3h, 33% (two steps); (iii) tetrazole, di-tert-
butyl diisopropylphosphoramidite, 1:1 CH2Cl2/THF, rt, 12h; (iv) H2O2, rt, 4h; (v) 2M NaOH, MeOH, rt, 8h, 48% (three steps); (vi) 4, PyBOP,
DIEA, CH2Cl2, rt, 6h, 15%; (vii) 1:1 TFA/CH2Cl2, rt, 4h, 77%.
poration of a benzimidazole group in the Ôlinker regionÕ
of the analogues (Table 1, Figs. 2 and 3). Agonism at the
S1P4 receptor type, however, has been increased by one
to two log orders in the transition of N-aryl amide to
benzimidazole functionality.
compounds 7a and 7b and the phosphate compounds
9a and 9b. Compounds with free alcohol head groups
were determined to be approximately two to three log
orders less potent on S1P4 than their phosphate counter-
parts for compounds 7a, 7b, 9a, 9b, 12, and 14.
Although relatively potent EC50 values were obtained
for the benzimidazole compounds on S1P1 and S1P5,
the compounds are very poorly efficacious at these
receptor types. With regard to stereochemistry, S-con-
figuration at the C2 position resulted in slightly in-
creased potency at S1P4 for both of the alcohol
In a preliminary attempt to uncover metabolically stable
S1P analogues, with regard to phosphatase activity, the
benzimidazole compounds were methylated at the 1 and
2 positions. The racemic 2-methyl phosphate 18 lost
potency at S1P4 by approximately one to two log
orders, as compared to 9a and 9b. The (1R,2R)-1-methyl
Table 1. EC50 values (nM) for S1P and benzimidazole analogues at S1P receptors determined by a [c-35S]GTP binding assaya
Compound
S1P1
S1P2
S1P3
S1P4
S1P5
S1P
7a
7b
9a
9b
12
14
18
20
2.2
na
na
na
na
na
na
na
1.2
na
na
na
na
na
na
na
37
1.7
2100 (pa)
330 (pa)
37 (pa)
21 (pa)
2900 (pa)
240 (pa)
1000 (pa)
4200
1600
48
2400 (pa)
390 (pa)
6.6 (pa)
8.2 (pa)
na
36
33,000
94
1200
77 (pa)
370 (pa)
a Values are means of three experiments (pa = partial agonist, na = not active).

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J. J. Clemens et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4903–4906
S1P2
S1P1
S1P3
S1P4
S1P5
120
120
100
120
100
120
100
120
100
100
80
60
40
20
0
80
60
40
20
0
80
60
40
20
0
80
60
40
20
0
80
60
40
20
0
–20
–20
–20
–20
–20
-13-12-11-10 -9 -8 -7 -6 -5 -4
-13-12-11-10 -9 -8 -7 -6 -5 -4
-13-12-11-10 -9 -8 -7 -6 -5 -4
-13-12-11-10 -9 -8 -7 -6 -5 -4
-13-12-11-10 -9 -8 -7 -6 -5 -4
Log [drug]
Log [drug]
Log [drug]
Log [drug]
Log [drug]
♦
•
18
S1P
9a
9b
14
Figure 2. [c-35S]GTP binding to HEK293T cell membranes in response to S1P and benzimidazole based S1P analogues. Each data point represents
the mean of three determinations.
NH2
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O
O-alkyl ester/N-alkyl
amide analogues
X
O
P
n
OH ·TFA
HO
O
X = O, NH
NH2
H
N
O
O
N-aryl amide
analogues
P
HO
·TFA
OH
O
n
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Figure 3. Structures of previously reported S1P analogues (see Ref. 8).
phosphate 14, derived from L-threonine, retained
approximately half of the potency (94nM) on S1P4 as
compared to the nonmethylated counterpart 9a (48nM).
To summarize, we have synthesized a series of S1P ana-
logues that incorporate a benzimidazole ring system in
the ÔlinkerÕ region of the pharmacophore. This structural
modification has resulted in the generation of highly po-
tent and fully efficacious S1P4 selective agonists. We
have determined a slight preference in potency for 2S-
configuration as well as a necessity for the phosphate
head group to obtain significant potency. We have also
demonstrated, with regard to potency, a reasonable tol-
eration of 1-methylation of the benzimidazole analogues
whereas 2-methylation furnished a drastic loss in agon-
ism. Our findings have helped to develop the SAR of
S1P on the S1P4 receptor and will serve as the basis
for additional studies along this route.
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T. L. Bioorg. Med. Chem. Lett. 2003, 13, 3401; (b) Im, D.
S.; Clemens, J.; Macdonald, T. L.; Lynch, K. R. Biochem-
istry 2001, 40, 14053.
9. Brief assay protocol: S1P was purchased from Biomol
(Plymouth Meeting, PA). [c-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, 5lg of membranes from the
appropriate receptor and G-protein expressing HEK293T
cells were incubated in 0.1mL of GTP-binding buffer (in
mM: HEPES 50, NaCl 100, MgCl2 5), pH7.5 containing
5lg saponin, 10lM GDP, 0.1nM [c-35S]GTP (1200Ci/
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.
Acknowledgements
This work was supported by grants from the NIH (NIG-
MS R01 GM067958 (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:
Spiegel, S.; Milstien, S. J. Biol. Chem. 2002, 277, 25851.
2. For a brief review on the S1P receptors, see: (a) Chun, J.;
Anliker, B. J. Biol. Chem. 2004, 279, 20555–20558; For a
review on S1P receptor nomenclature, see: (a) Chun, J.;