Synthesis, pharmacology, and cell biology of sn-2-aminooxy analogues of lysophosphatidic acid

Article abstract of DOI:10.1021/ol7030747

An efficient enantioselective synthesis of sn-2-aminooxy (AO) analogues of lysophosphatidic acid (LPA) that possess palmitoyl and oleoyl acyl chains Is presented. Both sn-2-AO LPA analogues are agonists for the LPA₁, LPA₂, and LPA

Full text of DOI:10.1021/ol7030747

ORGANIC
LETTERS
2008
Vol. 10, No. 6
1111-1114
Synthesis, Pharmacology, and Cell
Biology of sn-2-Aminooxy Analogues of
Lysophosphatidic Acid
Joanna Gajewiak,† Ryoko Tsukahara,‡ Yuko Fujiwara,‡ Gabor Tigyi,‡ and
Glenn D. Prestwich*,†
Department of Medicinal Chemistry, The UniVersity of Utah, 419 Wakara Way,
Suite 205, Salt Lake City, Utah, 84108-1257, and Department of Physiology,
College of Medicine, UniVersity of Tennessee Health Science Center,
Memphis, Tennessee 38163
gprestwich@pharm.utah.edu
Received December 20, 2007
ABSTRACT
An efficient enantioselective synthesis of sn-2-aminooxy (AO) analogues of lysophosphatidic acid (LPA) that possess palmitoyl and oleoyl
acyl chains is presented. Both sn-2-AO LPA analogues are agonists for the LPA1, LPA2, and LPA4 G-protein-coupled receptors, but antagonists
for the LPA3 receptor and inhibitors of autotaxin (ATX). Moreover, both analogues stimulate migration of intestinal epithelial cells in a scratch
wound assay.
Lysophosphatidic acid (1- or 2-acyl-sn-glycerol 3-phosphate,
LPA) is a deceptively simple ligand that elicits a rich palette
of biological responses, including platelet aggregation,
promotion of cell survival, and cell migration.1,2 The most
important source of LPA is the action of lysophospholipase
D (lysoPLD), or autotaxin (ATX), on lysophosphatidyl
choline (LPC).3 ATX is one of the 40 most upregulated genes
in invasive cancers4 and has been implicated in cell motility
and tumor invasion, metastasis, and neovascularization.3 LPA
signals through the activation of specific receptors, which
in turn leads to distinct cellular events depending on the
receptor subtype expressed by the targeted cell. Cell surface
LPA receptors belong to the membrane G protein-coupled
receptors (GPCRs) protein family, and five mammalian LPA
GPCRs have been characterized: LPA1, LPA2, LPA3, LPA4,
and LPA5.1,5-8
Figure 1. Structures of LPA, OMPT, HE-LPA, and SAP.
† The University of Utah.
‡ University of Tennessee.
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Modification of LPA may involve either the phosphate
head group, the glycerol backbone, or the acyl groups,
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10.1021/ol7030747 CCC: $40.75
© 2008 American Chemical Society
Published on Web 02/20/2008

Scheme 1. Synthesis of sn-2-AO-LPA Intermediates 9a and 9b
leading to many changes in biological activity.9-12 Only a
strongly nucleophilic aminooxy (AO) group at the sn-2
position. AO compounds are frequently employed as potent
inhibitors of pyridoxal-5-phosphate-dependent enzymes, such
as aminotransferases, serine hydroxymethyltransferase, ty-
rosine decarboxylase, cystationase, and ornithine decarbox-
ylase, whereby the aminooxy moiety forms a stable oxime
with the aldehyde group present on the cofactor.18-22 AO
analogues have also been demonstrated to function in Vitro
as potent antimalarial agents.23
We used the Mitsunobu reaction to introduce the AO
functionality in stereocontrolled manner using straightforward
protection and deprotection steps, thus elaborating an efficient
synthetic approach to produce enantiomerically pure sn-2
AO-LPA analogues. Herein we describe the asymmetric total
syntheses of two sn-2-AO LPA analogues, as well as
evaluation in pharmacological, biochemical, and cell biologi-
cal assays that reveal unexpected agonist and antagonist
activities.
few analogues of LPA feature modifications only at the sn-2
position (Figure 1). For example, OMPT (1-oleoyl-2-O-
methyl-rac-glycerophosphothioate), in either the racemic13
or optically pure14 forms, are potent agonists for the LPA3
receptor. The HE-LPA (hydroxyethoxy-LPA) analogues15 are
modest agonists for LPA3 with 10-fold lower potency than
the parent oleoyl LPA. Finally, the sn-2-F LPA analogs16
show relatively weak agonist activity for all LPA receptor
isoforms.
Recently, series of novel cytotoxic phospholipids, LPA-
like serine amide phosphates (SAPs) were synthesized and
demonstrated to be low micromolar inhibitors of prostate
tumor cell proliferation.17 The key element of this structure
was the presence of a primary amine at the sn-2 position.
Rather than introduce a strongly basic group into the LPA
structure, our approach was to introduce the less basic but
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Synthesis. The synthesis of sn-2-AO LPA started with
benzylation of (S)-solketal (1) (Scheme 1), followed by
removal of the isopropylidene with an acidic ion-exchange
resin,24,25 to give intermediate diol 2. Next, TBDMS was
introduced26,27 at the primary hydroxyl group at 0-3 °C to
produce precursor 3, which was transformed Via a Mitsunobu
reaction at 0 °C28,29 into phthalimide derivative 4 with clean
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Org. Lett., Vol. 10, No. 6, 2008

Table 1. Pharmacological Characterization of 12a and 12b in LPA GPCR Assays
LPA1 LPA2
LPA3
LPA4
EC50 [nM]
IC50 [nM]
(Inhib. %)b
EC50 [nM]
IC50 [nM]
(Inhib. %)
EC50 [nM]
IC50 [nM]
(Inhib. %)
EC50 [nM]
(Emax)
IC50 [nM]
(Inhib. %)
a
a
a
a
compd
(Emax
)
(Emax
)
(Emax
)
12a
>4630
(57.3)
>7440
(51.2)
NEc
2450
(68.8)
382
NE
NE
NE
2670
(63.1)
56
>10800
(45.8)
1880
NE
NE
12b
NE
NE
(104)
(45.9)
(86.8)
a Emax ) maximal efficacy of compound/maximal efficacy of LPA 18:1 × 100. b Inhib. % ) % maximal inhibition of the response to 200 nM LPA 18:1
c NE ) no effect was shown at the highest concentration (30 µM) tested.
inversion of configuration at the stereogenic center. Hydra-
zine monohydrate treatment of compound 429 afforded the
aminooxy intermediate 5 in quantitative yield. The enantio-
meric purity of the aminooxy compound was verified by
conversion to the (R)-Mosher amide;30 19F NMR showed that
intermediate 5 to have >95% enantiomeric purity. Next, the
aminooxy group of 5 was reprotected with Boc to produce
derivative 6, and the benzyl ether was hydrogenolyzed to
yield alcohol 7 in over 95% yield. Compound 7 was
converted to the palmitoyl (8a) and oleoyl (8b) esters using
carbodiimide chemistry, and deprotection of the silyl ether
with Bu4NF26 in THF smoothly produced intermediates 9a
and 9b in high yields.
by addition of a wet CH2Cl2 (0.5% water) with stirring for
45 min. The crude compounds were then dried in Vacuo,
and 10% aq. methanol was added, which resulted in
simultaneous deprotection of Boc group and the phosphate
methyl esters. The desired compounds 12a and 12b (Scheme
2) were obtained in homogeneous form in good yields after
filtration of each methanol solution through a Whatman
PTFE Syringe Filter.
Pharmacology of AO-LPA Analogues on LPA Recep-
tors. The pharmacological properties of AO-LPA analogues
12a and 12b were evaluated on LPA1-3 GPCRs in the
McArtl rat hepatoma cell line RH7777, and on LPA4 in CHO
cells. Table 1 summarizes these data, which show that both
palmitoyl and oleoyl sn-2-AO LPA analogues are agonists
for the LPA1, LPA2, and LPA4 G-protein-coupled receptors
but are antagonists for the LPA3 receptor. The oleoyl
analogue 12b is the most potent (382 nM) and also a full
agonist for LPA2. This analogue is also the most potent (56
Scheme 2. Conversion of Intermediates to sn-2-AO-LPA
Analogues 12a and 12b
In attempting to remove both methyl and Boc protecting
groups, neither an excess of TMSBr nor TMSBr followed
by TFA, nor anhydrous HCl gave any desired product.
Finally, each ester 11a and 11b was treated with BSTFA
(N,O-bis(trimethylsilyl)trifluoroacetamide)32 and TMSBr for
1 h under strict anhydrous condition in CH2Cl2, followed
Figure 2. Compounds 12a and 12b promote cell migration in a
scratch wound assay using IEC-6 cells. The basal rate of cell
motility was measured in the presence of 0.1% BSA vehicle for
stabilizing the LPA or LPA analogue, and the maximal LPA-
induced response was evoked by treatment with 100 nM LPA. The
bars represent the mean rate of cell migration in scratch wounds
treated with different concentrations of 12a and 12b. The results
represent the rate of migration over an 8 h period and the mean of
8 experiments.
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Org. Lett., Vol. 10, No. 6, 2008
1113

nM) partial antagonist for LPA3. Both 12a and 12b inhibit
recombinant ATX activity at 10 µM with potencies similar
to those of LPA itself (see Supporting information).
Activity of AO-LPA Analogues on Cell Migration. The
AO-LPA analogues 12a and 12b enhanced rate cell migration
in IEC-6 human intestinal endothelial cells in a scratch
wound assay (Figure 2). In this assay, the rate of cell
migration is measured by the time it takes for the cells to
close a gap in the cell monolayer created by “scratching”
the monolayer with a pipet tip. This effect is congruent with
the agonist activity toward the LPA2 receptor, which is
coupled to the Rho/Rac1 small GTPases and augments cell
migration and cell survival.33
profound effects on the receptor pharmacology and cell
biology of the analogues. Instead of the pan-receptor agonist
activity exhibited by LPA, 12a and 12b are weak agonists
for LPA1 and LPA4, strong agonists for LPA2, and antago-
nists for the LPA3. This mixed profile isoform selectivity
makes them useful research tools for cell biology. Moreover,
their ability to enhance migration of intestinal epithelial cells
in Vitro suggests therapeutic potential for repair of the
gastrointestinal epithelium in ViVo.
Acknowledgment. This work was supported by the
National Institutes of Health (NS29632 to G.D.P. and
HL61469 and CA921160 to G.T.)
The replacement of the sn-2 hydroxy group of LPA with
the more nucleophilic aminooxy function afforded AO-LPA
analogues 12a and 12b. This small structural change has
Supporting Information Available: Experimental de-
tailes for the synthesis and characterization of new com-
pounds and protocols for biological assays. This material is
available free of charge via the Internet at http://pubs.acs.org
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