Synthesis of Migration-Resistant Hydroxyethoxy Analogues of Lysophosphatidic Acid

Article abstract of DOI:10.1021/ol0358758

(Matrix presented) The susceptibility of lysophosphatidic acid (LPA) to intramolecular acyl migration impedes the determination of specific receptor activation by the sn-1 and sn-2 LPA regloisomers. An efficient enantioselective synthesis of hydroxyethoxy

Full text of DOI:10.1021/ol0358758

ORGANIC
LETTERS
2003
Vol. 5, No. 24
4685-4688
Synthesis of Migration-Resistant
Hydroxyethoxy Analogues of
Lysophosphatidic Acid
Lian Qian,† Yong Xu,† Hiroyuki Arai,‡ Junken Aoki,‡ Thomas M. McIntyre,§ and
,†
Glenn D. Prestwich*
Department of Medicinal Chemistry, The UniVersity of Utah, 419 Wakara Way,
Suite 205, Salt Lake City, Utah 84108-1257, Graduate School of Pharmaceutical
Sciences, The UniVersity of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan,
and Program in Human Molecular Biology and Genetics, The UniVersity of Utah,
Salt Lake City, Utah 84112-5330
gprestwich@pharm.utah.edu
Received September 25, 2003
ABSTRACT
The susceptibility of lysophosphatidic acid (LPA) to intramolecular acyl migration impedes the determination of specific receptor activation
by the sn-1 and sn-2 LPA regioisomers. An efficient enantioselective synthesis of hydroxyethoxy (HE)-substituted analogues of sn-1-acyl and
2-acyl LPA derivatives that possess palmitoyl and oleoyl chains is described. While the palmitoyl derivatives fail to activate calcium release
in cells transfected with LPA2 or LPA3 G-protein-coupled receptors, the LPA3 receptor is activated by both 1-HE and 2-HE oleoyl LPA analogues
with a potency 10-fold lower than that of the parent oleoyl LPA.
Lysophosphatidic acid (LPA) is part of a family of bioactive
phospholipids with the general form of 1- or 2-O-acyl-sn-
glycero-3-phosphate (sn-1 or sn-2 LPA) that mediate a
variety of biologic effects.1,2 LPA induces cell proliferation,
morphological changes, and has been shown to be involved
in many physiological and pathological processes including
neurogenesis,3 myelination, angiogenesis,4 wound healing,5
and cancer progression.6,7
cerebrospinal fluid. LPA is present at elevated levels,
however, in the ascites of ovarian cancer patients, and may
thus contribute to the progression of human cancer.8 While
the sn-1 LPA acyl regioisomer is preferentially produced in
platelets, it appears that the sn-2 isomer is also found in
(3) Chun, J. Crit. ReV. Neurobiol. 1999, 13, 151-168.
(4) Sugiura, T.; Nakane, S.; Kishimoto, S.; Waku, K.; Yoshioka, Y.;
Tokumura, A. J. Lipid Res. 2002, 43, 2049-2055.
(5) Sturm, A.; Dignass, A. U. Biochim. Biophys. Acta 2002, 1582, 282-
288.
Normally, LPA is present in serum at low levels and is
not detectable in platelet-poor plasma, whole blood, or
(6) Fang, X.; Gaudette, D.; Furui, T.; Mao, M.; Estrella, V.; Eder, A.;
Pustilnik, T.; Sasagawa, T.; Lapushin, R.; Yu, S.; Jaffe, R. B.; Wiener, J.
R.; Erickson, J. R.; Mills, G. B. Ann. N.Y. Acad. Sci. 2000, 905, 188-208.
(7) Mills, G. B.; Moolenaar, W. H. Nat. ReV. Cancer 2003, 3, 582-
591.
(8) Erickson, J. R.; Hasegawa, Y.; Fang, X.; Eder, A.; Mao, M.; Furui,
T.; Aoki, J.; Morris, A.; Mills, G. B. Prostaglandins Other Lipid Mediators
2001, 64, 63-81.
† Department of Medicinal Chemistry, The University of Utah.
‡ The University of Tokyo.
§ Program in Human Molecular Biology and Genetics, The University
of Utah.
(1) Moolenaar, W. H. J. Biol. Chem. 1995, 270, 12949-12952.
(2) Feng, L.; Mills, G. B.; Prestwich, G. D. Expert Opin. Ther. Pat. 2003,
in press.
10.1021/ol0358758 CCC: $25.00 © 2003 American Chemical Society
Published on Web 10/30/2003

ascites.9 Therefore, levels of sn-2 LPA seem to be associated
with the initiation and progression of ovarian cancer.10
However, sn-2 LPA is not stable under physiological
conditions; it is readily converted under both acidic and basic
conditions to an equilibrium mixture of sn-1 and sn-2 LPA
isomers via intramolecular acyl chain migration (Scheme 1,
We replaced the hydroxyl group with a hydroxyethoxy (HE)
group to render acyl migration unfavorable. The advantage
of this design is 2-fold. First, the HE group replacement will
maintain a functional “lyso-like” structure, since initial
structure-activity relationship (SAR) studies19 have revealed
that a hydroxyl group, an alkyl of more than 12 carbon atoms,
and a phosphate monoester are required for the biological
activities of LPA. Second, the HE group creates an unfavor-
able intermediate for the intramolecular acyl20 migration that
compromises the stereochemistry by regioisomerization
(Scheme 1, bottom).
Scheme 1. Favorable Intermediate for Acyl Migration for LPA
(top) and Unfavorable Intermediate for Migration in HE-LPA
Analogues (bottom)
Traditionally, GPCRs have shown a preference for the
naturally occurring enantiomer of their cognate ligands.
However, our preliminary biological results have demon-
strated that the unnatural D (2S) stereoisomers of some
O-methylated LPA analogues (OMPT)21 are more active than
naturally occurring L (2R) enantiomorphs (Qian, Xu, Mills,
Aoki, and Prestwich, submitted for publication). On the basis
of these results, we synthesized the (2S) enantiomers of sn-1
and sn-2 acyl HE-LPA as our desired nonmigrating LPA
analogues using an efficient and stereoselective chemical
synthetic route.
As shown in Scheme 2, regiospecific and stereospecific
top).11 The lability of the acyl group in a 2-acyl-sn-glycerol-
3-phosphate impedes structure-activity studies of individual
LPA species.
Scheme 2. Synthesis of 1-Acyl, 2-HE LPA Analoguesa
LPA signaling occurs through specific cell surface recep-
tors of the endothelial cell differentiation gene (Edg) family
of seven-transmembrane domain G-protein-coupled receptors
(GPCR), which includes Edg-2/LPA1, Edg-4/LPA2, and Edg-
7/LPA3.12 Recently, an additional LPA receptor, p2y9/
GPR23/LPA4, only distantly related to the Edg receptors and
more closely related to the purinergic receptors, has been
identified.13 However, the function of particular receptors
in the mammalian system and the molecular mechanism of
LPA actions are still subjects of intensive investigation.14
Previously, we described the enantioselective synthesis and
biological activities of a variety of fluorinated LPA analogues
as metabolically stabilized ligands for LPA receptors.15-18
Herein, we report an alternative approach to create LPA
analogues with improved stability as useful molecular tools.
a Reagents and conditions: (a) PMBOH, DIBAL, CH2Cl2, 51%;
(b) TBDMSCl, DMAP, TEA, CH2Cl2, 78%; (c) NaH, TBAI,
BrCH2CH2OTHP, DMF, 56%; (d) TBAF, THF, 95%; (e) Oleic acid
(palmitic acid), DCC, DMAP, CH2Cl2, 82%; (f) DDQ, CH2Cl2,
66%; (g) (OMe)2P(O)Cl, t-BuOK, 75%; (h) TMSBr, MeOH/H2O,
95%.
(9) Mills, G. B.; Eder, A.; Fang, X.; Hasegawa, Y.; Mao, M.; Lu, Y.;
Tanyi, J.; Tabassam, F. H.; Wiener, J.; Lapushin, R.; Yu, S.; Parrott, J. A.;
Compton, T.; Tribley, W.; Fishman, D.; Stack, M. S.; Gaudette, D.; Jaffe,
R.; Furui, T.; Aoki, J.; Erickson, J. R. Cancer Treat. Res. 2002, 107, 259-
283.
ring opening of (S)-glycidol22 by 4-methoxybenzyl alcohol
(PMB-OH), using diisobutylaluminum hydride (DIBAL),
generated the PMB protected glycerol (1). After selective
(10) Fang, X.; Schummer, M.; Mao, M.; Yu, S.; Tabassam, F. H.; Swaby,
R.; Hasegawa, Y.; Tanyi, J. L.; LaPushin, R.; Eder, A.; Jaffe, R.; Erickson,
J.; Mills, G. B. Biochim. Biophys. Acta 2002, 1582, 257-264.
(11) Chevallier, J.; Sakai, N.; Robert, F.; Kobayashi, T.; Gruenberg, J.;
Matile, S. Org. Lett. 2000, 2, 1859-1861.
(12) Contos, J. J.; Ishii, I.; Chun, J. Mol. Pharmacol. 2000, 58, 1188-
1196.
(19) Lynch, K. R.; Macdonald, T. L. Biochim. Biophys. Acta 2002, 1582,
289-294.
(13) Noguchi, K.; Ishii, S.; Shimizu, T. J. Biol. Chem. 2003, 278, 25600-
25606.
(20) Yokoyama, K.; Baker, D. L.; Virag, T.; Liliom, K.; Byun, H. S.;
Tigyi, G.; Bittman, R. Biochim. Biophys. Acta 2002, 1582, 295-308.
(21) Hasegawa, Y.; Erickson, J. R.; Goddard, G. J.; Yu, S.; Liu, S.;
Cheng, K. W.; Eder, A.; Bandoh, K.; Aoki, J.; Jarosz, R.; Schrier, A. D.;
Lynch, K. R.; Mills, G. B.; Fang, X. J. Biol. Chem. 2003, 278, 11962-
11969.
(14) Bandoh, K.; Aoki, J.; Taira, A.; Tsujimoto, M.; Arai, H.; Inoue, K.
FEBS Lett. 2000, 478, 159-165.
(15) Xu, Y.; Prestwich, G. D. J. Org. Chem. 2002, 67, 7158-7161.
(16) Xu, Y.; Prestwich, G. D. Org. Lett. 2002, 4, 4021-4024.
(17) Xu, Y.; Qian, L.; Prestwich, G. D. J. Org. Chem. 2003, 68, 5320-
5330.
(22) Erukulla, R. K.; Byun, H. S.; Locke, D. C.; Bittman, R. J. Chem.
Soc., Perkin. Trans. 1 1995, 11367-11597.
(18) Xu, Y.; Qian, L.; Prestwich, G. D. Org. Lett. 2003, 5, 2267-2270.
4686
Org. Lett., Vol. 5, No. 24, 2003

silylation at the primary alcohol by tert-butyldimethylsilyl
(TBDMS) chloride,23 the secondary alcohol 2 was alkylated
with 2-(2-bromoethoxy)tetrahydro-2H-pyran in the presence
of NaH and tetrabutylammonium iodide (TBAI) in good
yield.24 The 1-TBDMS ether 3 was then deprotected with
tetra(n-butyl)ammonium fluoride (TBAF) in THF to give
alcohol 4, which was esterified with oleic acid or palmitic
acid with use of DCC and DMAP to produce esters 5a and
5b, respectively. Oxidative removal of the PMB group with
2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) produced cor-
responding alcohols 6a and 6b. Phosphorylation with di-
methyl chlorophosphate in the presence of t-BuOK gave
good yields of protected phosphates 7a and 7b. The
nonreductive deprotection of dimethyl phosphates with
bromotrimethylsilane18 was compatible with the unsaturated
acyl chains. The trace amount of acid generated during
workup (adding MeOH/H2O) resulted in elimination of the
tetrahydropyranyl group (THP) and generation of the sn-1-
acyl 2-HE-modified LPA analogues 8a and 8b.
2-TBDMS ether was further deprotected with TBAF-3H2O
in THF to give alcohol 13. Neutralization of TBAF with
acetic acid allowed the desilylation of the secondary alcohol
without migration of the phosphate. After DCC-promoted
esterification and deprotection of the phosphate with TMSBr,
sn-2 LPA analogues 15a and 15b were obtained in high
yields.
Each of the nonmigrating LPA analogues 8a, 8b, 15a, and
15b was incubated in Tris-HCl buffer (pH 8.0) at 37 °C for
1
24 h. No migration or decomposition was observed by H
NMR spectroscopy, which demonstrates the good chemical
stability of those LPA analogues under biological assay
conditions.
The synthetic LPA analogues were evaluated for their
ability to activate transmembrane LPA receptors and nuclear
PPARγ. First, the LPA analogues were tested in insect Sf9
cells expressing LPA2 or LPA3 receptors,26 and in a cell
motility assay with rat hepatoma Rh7777 cells that express
LPA1.27 None of the analogues tested activated LPA1 (Figure
1), and only modest calcium release was induced by
The strategy for the synthesis of the nonmigrating sn-2
1-HE-LPA analogues (Scheme 3) was similar to that above
Scheme 3. Synthesis of 2-Acyl, 1-HE LPA Analoguesa
a Reagents and conditions: (a) THPOCH2CH2OH, DIBAL,
CH2Cl2, 50%; (b) TBDMSCl, imidazole, DMF, 91%; (c) HF-Py/
Py, THF, 58%; (d) (OMe)2P(O)Cl, 1-methylimidazole, 87%; (e)
TBAF, AcOH, THF, 76%; (f) oleic acid (palmitic acid), DCC,
DMAP, CH2Cl2, 85%; (g) TMSBr, MeOH/H2O, 95%.
Figure 1. Cell motility assay by activation of LPA1 in transfected
Rh7777 cells: (b) LPA (1-oleoyl); (2) 8a (2-HE, 1-oleoyl,); (4)
8b (2-HE, 1-palmitoyl); (9) 15a (1-HE, 2-oleoyl); and (0) 15b
(1-HE, 2-palmitoyl).
activation of LPA2 by any of the compounds (Figure 2).
However, the oleoyl HE-LPA analogues 8a and 15a activated
LPA3 with potency 30-fold less than that of 1-oleoyl LPA.
8a and 15a also showed over 100-fold higher potency than
the two palmitoyl HE-LPA analogues 8b and 15b, confirm-
ing that LPA3 markedly prefers ligands with an unsaturated
fatty-acid ester. Interestingly, little difference was observed
between the 1-HE and 2-HE oleoyl regioisomers 8a and 15a
for LPA3 activation, which can be attributed to the extended
HE group.
for sn-1 2-HE-LPA. To obtain the (2S) enantiomer, (R)-
glycidol was regioselectively ring-opened and alkylated with
2-(2-bromoethoxy)tetrahydro-2H-pyran to give diol 9, fol-
lowed by bis-silylation to 10. Selective deprotection of bis-
TBDMS ether 10 with 6.0 equiv of pyridinium hydrofluoride
(HF-Py)25 resulted in 58% yield of alcohol 11 after 18 h at
room temperature. The optimal amount of HF-Py was crucial
to the reaction, since an excess would cause deprotection of
both TBDMS groups. Phosphorylation of 11 with use of
dimethyl chlorophosphate in the presence of N-methylimi-
dazole gave protected phosphate in 87% yield.21 Use of
t-BuOK as base for this reaction gave only 10% yield. The
In addition, the LPA analogues were evaluated in a nuclear
reporter assay in which monocytic cells were transfected with
(26) Bandoh, K.; Aoki, J.; Hosono, H.; Kobayashi, S.; Kobayashi, T.;
Murakami-Murofushi, K.; Tsujimoto, M.; Arai, H.; Inoue, K. J. Biol. Chem.
1999, 274, 27776-27785.
(27) Umezu-Goto, M.; Kishi, Y.; Taira, A.; Hama, K.; Dohmae, N.;
Takio, K.; Yamori, T.; Mills, G. B.; Inoue, K.; Aoki, J.; Arai, H. J. Cell
Biol. 2002, 158, 227-233.
(23) Chen, J.; Prestwich, G. D. J. Org. Chem. 1998, 63, 430-431.
(24) Golec, J. M. C.; Hedgecock, C. J. R.; Murdoch, R.; Tully, W. R.
Tetrahedron Lett. 1992, 33, 551-554.
(25) Nazare, M.; Waldmann, H. Angew. Chem., Int. Ed. 2000, 39, 1125-
1128.
Org. Lett., Vol. 5, No. 24, 2003
4687

Figure 3. HE-LPA analogues and natural LPA activate the nuclear
transcription factor PPARγ: ROSI, rosiglitazone; LPA (1-oleoyl);
8a (2-HE, 1-oleoyl); 8b (2-HE, 1-palmitoyl); and 15b (1-HE,
2-palmitoyl).
homologues consistent with previous results.28 Moreover, the
HE-LPA derivatives are expected to be less susceptible to
LPA acyl transferase activity.29
In summary, we have established a concise and efficient
approach for synthesizing HE-substituted nonmigrating LPA
analogues. We have demonstrated that 1-HE and 2-HE oleoyl
LPA analogues 8a and 15a, at e1 µM, triggered cellular
responses through the LPA3 but not the LPA1 or LPA2
receptors. Neither 8a nor 15a were LPA1 receptor agonists
at above 1 µM. Thus, oleoyl HE-LPA analogues are useful
probes for the dissection of LPA signaling events in complex
mammalian systems. Further application of these LPA
analogues is expected to provide additional insight into the
complex biology of LPA.
Figure 2. Calcium release by activation of LPA2 (top) and LPA3
(bottom) in transfected Sf9 cells: (b) LPA (1-oleoyl); (2) 8a (2-
HE, 1-oleoyl); (4) 8b (2-HE, 1-palmitoyl); (9) 15a (1-HE,
2-oleoyl); and (0) 15b (1-HE, 2-palmitoyl).
Acknowledgment. We thank the Utah Centers of Excel-
lence Program, the Human Frontier Science Program
(RG0073-2000-B), and the NIH (NS29632 to G.D.P. and
HL070231 to Dr. T. M. McIntyre (UUtah)) for financial
support of this work.
a luciferase construct activated by a PPARγ nuclear receptor
response element (Figure 3). In this assay, three of the four
analogues, as well as sn-1-oleoyl-LPA, were tested. We
found that the nuclear transcription factor PPARγ was
activated by both the sn-1 and sn-2 HE-LPA analogues, and
did not show a preference for an unsaturated acyl residue.17,28
PPARγ, therefore, responds to a wide range of LPA
Supporting Information Available: Experimental pro-
cedures and characterization for all new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL0358758
(28) McIntyre, T. M.; Pontsler, A. V.; Silva, A. R.; St Hilaire, A.; Xu,
Y.; Hinshaw, J. C.; Zimmerman, G. A.; Hama, K.; Aoki, J.; Arai, H.;
Prestwich, G. D. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 131-136.
(29) Yamashita, A.; Kawagishi, N.; Miyashita, T.; Nagatsuka, T.; Sugiura,
T.; Kume, K.; Shimizu, T.; Waku, K. J. Biol. Chem. 2001, 276, 26745-
26752.
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