Synthesis of monofluorinated analogues of lysophosphatidic acid

Article abstract of DOI:10.1021/jo020729l

Lysophosphatidic acid (LPA, 1- or 2-acyl-sn-glycerol 3-phosphate) displays an intriguing cell biology that is mediated via interactions both with G-protein coupled seven transmembrane receptors and with the nuclear hormone receptor PPARγ. Synthesis and bi

Full text of DOI:10.1021/jo020729l

Syn th esis of Mon oflu or in a ted An a logu es of Lysop h osp h a tid ic Acid
Yong Xu, Lian Qian, and Glenn D. Prestwich*
Department of Medicinal Chemistry and The Center for Cell Signaling, The University of Utah,
419 Wakara Way, Suite 205, Salt Lake City, Utah 84108-1257
gprestwich@pharm.utah.edu
Received December 9, 2002
Lysophosphatidic acid (LPA, 1- or 2-acyl-sn-glycerol 3-phosphate) displays an intriguing cell biology
that is mediated via interactions both with G-protein coupled seven transmembrane receptors and
with the nuclear hormone receptor PPARγ. Synthesis and biological activities of fluorinated
analogues of LPA are still relatively unknown. In an effort to identify receptor-selective LPA
analogues and to document in detail the structure-activity relationships of fluorinated LPA
isosteres, we describe a series of monofluorinated LPA analogues in which either the sn-1 or the
sn-2 hydroxy group was replaced by fluorine, or the bridging oxygen in the monophosphate was
replaced by an R-monofluoromethylene (-CHF-) moiety. The sn-1 or sn-2 monofluorinated LPA
analogues were enantiospecifically prepared from chiral protected glycerol synthons, and the
R-monofluoromethylene-substituted LPA analogues were prepared from a racemic epoxide with
use of a hydrolytic kinetic resolution. The sn-2 and sn-1 fluoro LPA analogues were unable to undergo
acyl migration, effectively “freezing” them in the sn-1-O-acyl or sn-2-O-acyl forms, respectively.
The R-monofluoromethylene LPA analogues were unique new nonhydrolyzable ligands with
surprising enantiospecific and receptor-specific biological readouts, with one compound showing a
1000-fold higher activity than native LPA for one receptor subtype.
In tr od u ction
Lysophosphatidic acid (LPA, 1- or 2-acyl-sn-glycerol
3-phosphate) (Figure 1, 4) is a naturally occurring
phospholipid with a deceptively simple structure. It has
received increasing attention due to the variety of
biological responses that it evokes, including platelet
aggregation, smooth-muscle contraction, changes in cell
morphology, and mitogenesis.1 LPA elicits these cellular
events via signal transduction cascades downstream of
its specific interactions with three G-protein-coupled
receptors (GPCR) belonging to the endothelial differen-
tiation gene (edg) family.2 These receptors recently have
been reclassified as LPA and sphingosine 1-phosphate
receptors. In particular, edg-2, edg-4, and edg-7 are now
the LPA-responsive receptors known as LPA1, LPA2, and
LPA3, respectively.3,4 Surprisingly, LPA has a completely
separate function as an intracellular lipid signal, based
on its recent identification as a naturally occurring ligand
for the nuclear hormone receptor PPARγ.5
F IGUR E 1. Metabolically stabilized monofluoro analogues
(1-3) and parent sn-1-O-acyl lysophosphatidic acid (4).
of ovarian cancer.7,8 Signaling by LPA and other lyso-
lipids is an important new direction for the development
of new diagnostics and therapeutics to treat this disease.9
Interestingly, the LPA found in serum and blood differs
in that the ascites LPA appear to be enriched in 2-acyl
LPA species.7,10 Studying this 2-acyl LPA isoform has
been difficult due to its chemical lability. Intramolecular
acyl chain migration, which is facilitated by both acidic
and basic conditions, affords an equilibrium mixture of
LPA is released by activated platelets and accumulates
in serum at low micromolar levels.6 Substantial amounts
of LPA also occur in the malignant ascites characteristic
(7) Xu, Y.; Gaudette, D. C.; Boynton, J . D.; Frankel, A.; Fang, X. J .;
Sharma, A.; Hurteau, J .; Casey, G.; Goodbody, A.; Mellors, A. Clin.
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Tigyi, G. J AMA, J . Am. Med. Assoc. 2002, 287, 3081-3082.
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Hinshaw, J .; Zimmerman, G.; Hama, K.; Aoki, J .; Arai, H.; Prestwich,
G. D. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 131-136.
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10.1021/jo020729l CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/31/2003
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J . Org. Chem. 2003, 68, 5320-5330

Monofluoro LPA Analogues
1-acyl- and 2-acyl-sn-glycerol-3-phosphates that favors
the 1-acyl isoforms in approximately an 85:15 ratio at
equilibrium.11 Thus, the instability of 2-acyl-sn-glycerol-
3-phosphate seriously compromises both isolation of
naturally occurring species and determination of the
activating ligand in structure-activity studies. Moreover,
since the principal route of regulation of LPA involves
its conversion to monoacylglycerol by lipid phosphate
phosphatases (LPPs),12-14 it has been difficult to evaluate
what molecular LPA species are active and at what level
in cells and tissues.
isosteric replacement of oxygen by monofluoromethyl-
ene27,28 in phosphate analogues confers metabolic stability
and important features for receptor binding. Many
monofluoromethylene-substituted phosphonate deriva-
tives have been studied25,29 as potential enzyme inhibitors
and as probes for the elucidation of biochemical processes.
Thus, we also describe herein a new approach to the
synthesis of R-monofluoromethylene-substituted phos-
pholipids, and we present the application of this approach
to the preparation of a series of phosphatase-resistant
LPA analogues.
The isosteric substitution of essential hydroxyl groups
by fluorine has been a mainstay of analogue design when
metabolic stability is desired. It is particularly favored
as a substituent when the presence of an electronegative
atom is sufficient for the interaction of the ligand with
the target protein.15,16 Indeed, the high electron density
gives rise to the ability of the fluorine substituent to act
as an acceptor in intra- and intermolecular hydrogen
bonds.17-19 These interactions can result in modified
binding to a receptor.20 Therefore, we have developed a
program to test the hypothesis that fluorinated analogues
of LPA, particularly with fluorine in the sn-1 or sn-2
position, might mimic LPA as a biological ligand. Such
mimics would not undergo intramolecular acyl chain
migration, and could thus be useful in defining the regio-
chemical selectivity of LPA receptors for the acyl position.
Resu lts a n d Discu ssion
The synthetic strategies of the target monofluoro
compounds 1-fluorodeoxy-2-acyl-sn-glycerol-3-phosphate
1 and 1-acyl-2-fluorodeoxy-sn-glycerol-3-phosphate 2 were
based on three design considerations. First, it was
necessary to install the fluorine prior to acylation to avoid
acyl chain migration during the synthesis. Second, we
required both the natural 2R and unnatural 2S enanti-
omers of each LPA analogue to test for enantiospecific
biological responses to 2-acyl or 2-fluoro stereoisomers.
Third, for ease of synthetic manipulations, the deprotec-
tion of the penultimate dimethyl phosphate with tri-
methylsilane bromide (TMSBr)
permit incorporation of unsaturated acyl chains as well
as to reveal the charged phosphate at the final step of
the synthesis.
The key step for the synthesis of 1 and 2 was the
stereoselective introduction of fluorine at the C-1 or C-2
position. Deoxyfluorination was accomplished by using
diethylaminosulfur trifluoride (DAST),32,33 an easily
handled reagent that stereoselectively replaces hydroxyl
with fluorine under mild conditions. Thus, 1-fluorodeoxy-
(2R)-acyl-sn-glycerol-3-phosphates 1a and lb were syn-
thesized from commercially available (R)-isopropylidene-
glycerol 5. Alcohol 5 was first phosphorylated with
dimethylphosphoryl chloride in the presence of t-BuOK
to give dimethyl phosphate 6 in 92% yield.34,35 Next,
phosphate 6 was converted to the 2-silyl ether 3-phos-
phate in three steps. Acetonide hydrolysis with pTsOH/
MeOH gave a crude diol, which was converted directly
to the bis-silyl ether 8 by treatment with tert-butyl-
dimethylsilyl (TBS) chloride and imidazole in anhydrous
DMF. The more labile primary TBS ether was then
selectively cleaved with use of pyridium-HF in pyridine-
30,31
was selected to
Phosphate monoesters are susceptible to hydrolysis by
the action of both general and specific phosphatases. In
particular, since LPP is a major source of LPA catabo-
lism,12 we sought methods to create phosphatase-
resistant LPA analogues. Phosphonates, in which the
bridging oxygen is replaced by a carbon have been
extensively investigated as phosphatase-stable phosphate
22,23
mimics.21 In one refinement of this strategy,
the
bridging oxygen can be replaced by a CF2 substituent.
This is attractive as the fluorine atoms reintroduce the
electronegativity that was lost due to the replacement
of the oxygen atom. Although the R,R-difluorophospho-
nates have been more widely investigated in the last two
decades, recent experimental and theoretical reports have
indicated that an R-fluorophosphonate would be a better
mimic for the phosphate.24-26 The isoelectronic and
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Brindley, D. N. J . Biol. Chem. 2000, 275, 27520-27530.
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J . Org. Chem, Vol. 68, No. 13, 2003 5321

Xu et al.
SCHEME 1. Syn th esis of En a n tiom er ic sn -1-F lu or o-2-O-a cyl LP A An a logu esa
a
The final LPA analogues in all schemes and figures are shown in the phosphoric acid form but are stored and used as mixed neutral
sodium salts.
THF at room temperature.36 By using an optimized
The 1-acyl-(2S)-fluorodeoxy-sn-glycerol-3-phosphates
2a and 2b were synthesized from (S)-isopropylideneg-
lycerol 13. As described above for diol 7, diol 14 was
prepared by phosphorylation with dimethylphosphoryl
chloride followed by acid hydrolysis. Initial unsuccessful
attempts involved acylation of the 1-position followed by
reaction with DAST at -78 °C to introduce the 2-fluoro
group; extensive side reactions led us to abandon this
short-cut. Instead, the primary alcohol was selectively
protected as the tert-butyldiphenylsilyl (TBDPS) ether.
Thus, treatment of diol 14 with the TBDPS chloride gave
the sn-1 TBDPS ether 15. Deoxyfluorination of 15 gave
good yields of the 2-fluorinated product 16. Deprotection
of ether 16 with TBAF in THF gave alcohol 17, which
was esterified with either oleic or palmitic acids as
described above to give the target protected LPA deriva-
tives 18a and 18b. Deprotection of the phosphotriester
with bromotrimethylsilane afforded the desired fluori-
nated LPA analogues 2a and 2b. Similarly, the enantio-
meric (2R)-2-fluorodeoxy LPA analogues 2c and 2d were
synthesized from (R)-isopropylideneglycerol 5.
selective deprotection, a 63% yield was obtained. Nucleo-
philic displacement of hydroxyl with DAST in anhydrous
CH2Cl2 gave the corresponding monofluorinated com-
pound 10, without affecting the 2-TBS ether. The 2-hy-
droxyl was unmasked with use of tetra-(n-butyl)ammo-
nium fluoride (TBAF) in THF; neutralization of TBAF
with acetic acid permitted desilylation without phosphate
migration.37 DCC-promoted esterification of 11 with
either oleic acid or palmitic acid provided good yields of
esters 12a and 12b, which were purified to >99%
homogeneity prior to deprotection. Finally, treatment of
each ester 12 with TMSBr and subsequent addition of
5% aq methanol provided the desired fluorinated LPA
analogues 1a and 1b in nearly quantitative yield. With
use of the same procedure, the (2S)-LPA analogue 1c was
obtained from (S)-isopropylideneglycerol 13 in the analo-
gous eight steps (5.6% overall yield).
We have found that the acid forms of these LPA
analogues can be labile during storage or when made as
stock solutions for biological evaluation. Thus, we have
adopted a standard protocol to obtain a stable sodium
salt form of each LPA analogue. For example, 1a was
dissolved in 1.0 M triethylammonium bicarbonate (TEAB)
buffer (pH 8.0) to give a slightly cloudy solution, which
was absorbed onto a sodium ion-exchange column (Dowex
50WX8-200 resin, neutral Na+ form). The desired mixed
neutral sodium salt of 1a was eluted with Nanopure
water. The product solution was lyophilized to give an
amorphous white powder, which was stored in solid form
at -80 °C. Aqueous or DMSO solutions of LPA analogues
were prepared and used within several days to minimize
hydrolysis or other decomposition.
Current approaches to the synthesis of monofluoro-
methylene phosphonates include electrophilic fluo-
rination (Selectorfluor, FN-(SO2Ph)2),38,39 nucleophilic
fluorination (DAST),40,41 Arbuzov reactions with fluoro-
halomethanes,42 transition metal catalyzed addition of
diethyl monofluoroiodomethyl-phosphonate to alkene,43,44
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Monofluoro LPA Analogues
SCHEME 2. Syn th esis of En a n tiom er ic sn -1-O-Acyl-2-flu or o LP A An a logu es
SCHEME 3. Syn th esis a n d Hyd r olytic Kin etic Resolu tion of F lu or op h osp h on a te Ep oxid e
and displacement of triflates or iodides by fluorosulfonyl-
alkyl-phosphonates.27 Recently, we have reported a facile
and practical method for the synthesis of enantiomeric
pure R,R-difluoromethylene phosphonate analogues of
LPA by hydrolytic kinetic resolution (HKR) of a racemic
epoxide.45 We selected this strategy for the preparation
of the R-monofluoromethylene phosphonate analogues 3.
Thus, as illustrated in Scheme 3, 1-fluoro-3,4-epoxy-
butylphosphonate 22 (IUPAC numbering) was prepared
by addition of iodofluoromethylene-phosphonate 20 to
allyl alcohol and subsequent base-induced cyclization of
the iodohydrin 21 to epoxide 22. The HKR reaction,46-48
using two enantiomeric cobalt salen complexes 23 as
catalysts, would be used for kinetic resolution of the
terminal epoxide of 22 to obtain enantiomerically en-
riched diols 24a and 24b. These diols in turn would be
mono-acylated to give the corresponding enantiomeric
R-monofluoromethylene phosphonate LPA analogues 3.
Scheme 3 shows the final synthetic route for these
analogues. First, iodomonofluoromethyl phosphonate 20
was prepared in good yield from commercially available
diethyl dibromofluoromethyl phosphonate 19 by tributyl-
phosphine reduction and iodine quench of the intermedi-
ate zinc species.44 Next, the tetrakis(triphenylphosphine)-
palladium-catalyzed addition of phosphonate 20 to allyl
(45) Xu, Y.; Prestwich, G. D. Org. Lett. 2002, 4, 4021-4024.
(46) Tokunaga, M.; Larrow, J . F.; Kakiuchi, F.; J acobsen, E. N.
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J . Org. Chem, Vol. 68, No. 13, 2003 5323

Xu et al.
SCHEME 5. Dia ster eoselective Syn th esis of
sn -1-O-Acyl r-F lu or om eth ylen ep h osp h on a te LP A
An a logu es
SCHEME 4. Con ver sion of Resolved
F lu or op h osp h on a te Diols to sn -1-O-Acyl
r-F lu or om eth ylen ep h osp h on a te An a logu es
diethyl phosphonates 26 with excess TMSBr (10.0 equiv)
for 8 h at room temperature.
Since we were unable to separate the diastereomeric
1-fluoro-3-hydroxyl isomers of compounds 24, 26, or
3, we selected an alternative approach to prepare a
diastereomerically enriched R-monofluorinated phospho-
nate. For this synthesis, (2S)-1,2,4-butanetriol 27 was
protected as the isopropylidene acetal and oxidized with
PDC to give aldehyde 28.50 The Pudovik reaction was
then employed to introduce the C-P bond.51 Thus, the
anion of diethyl phosphite was added to aldehyde 28 at
-20 °C to give two chromatographically inseparable
R-hydroxyl phosphonates 29 in modest overall yield. This
addition reaction occurred without diastereoselectivity,
since two single sharp resonances at 25.37 and 24.47 ppm
of equal intensity were observed in the 31P NMR spec-
trum. This diastereomeric mixture was treated directly
with DAST, which gave a pair of diastereomers in a 6.3:1
ratio as determined by both 31P NMR and 19F NMR in
modest yield. Recently, Berkowitz also reported that
DAST-mediated conversion of a nonbenzylic, secondary
(R-hydroxyl)phosphonate to the (R-monofluoro)phospho-
nate proceeded with good diastereoselectivity.40 After
deprotection by acid hydrolysis and selective esterifica-
tion, phosphonate 26a a was obtained in >89% de.
Finally, TMSBr deprotection gave the final product 3a a
showing >89% de. Since no reference materials were
available, and NMR methods failed to define the relative
geometries of the C-H bonds at C-1 and C-3, we cannot
assign the absolute configuration at C-1 to this predomi-
nant stereoisomer at this time.
The preparation of receptor-specific agonists and an-
tagonists for LPA receptors is an active area of ligand
design. Structure-activity studies have demonstrated
that analogues 31and 32 (see Figure 2), lacking the
2-hydroxy group and structurally different analogues,
such as the N-palmitoylserine and N-palmitoyltyrosine
phosphoric acids 33 and 34, are potent competitive
antagonists of LPA receptor function in Xenopus oo-
cytes.52 However, as yet, a comprehensive analysis of
fluorinated LPA analogues as selective agonists or an-
tagonists for individual LPA receptors has not yet been
reported. The monofluorinated analogues described herein,
alcohol in hexane gave the corresponding iodohydrin
21 in 79% yield. Treatment of the iodohydrin with dilute
K2CO3/MeOH solution for 5 min at room temperature
provided the desired epoxide 22 in good yield (72%).49 It
is important to note that the racemic epoxide is also a
mixture of fluorine epimers at C-1, as demonstrated by
the two equal multiplets in the 19F NMR spectra of this
and subsequent intermediates. Next, reaction of racemic
epoxide 22 with 0.45 equiv of H2O in a minimum volume
of THF, in the presence of 1.0 mol % of (R,R)-23-OAc,
gave diol 24a in 90% ee and 73% isolated yield. Similarly,
catalyst (S,S)-23-OAc provided the opposite configuration
of diol 24b in 89% ee and 90% yield. The epoxide and
diol were readily separated by flash chromatography,
providing a further extension of the scope of the HKR
process, which was previously employed to prepare the
difluoromethylene phosphonates.45 Each diol was isolated
as an inseparable, equimolar mixture of two diastereo-
mers epimeric at C-1. For initial assessment of biological
activity, the separation of this epimeric mixture at the
C-1 phosphonate methylene was not required.
Regioselective acylation of the primary hydroxyl of
diols 24 was readily accomplished (Scheme 4). Note that
the numbering employed henceforth for the phosphonate
LPA analogues 24, 25, 26, and 3 employs the sn-glycerol
nomenclature for clarity of comparison with other LPA
derivatives. Thus, treatment of 24a with 0.95 equiv of
oleic acid and 1.2 equiv of DCC and DMAP in CH2Cl2 at
0 °C gave 26a a in 42% yield, following chromatography
to remove a small amount of diester. The corresponding
palmitate 26a b was similarly produced, as were the
enantiomeric oleate 26ba and palmitate 26bb. Finally,
LPA analogues 3 were obtained by dealkylation of the
(50) Zheng, X. P.; Nair, V. Tetrahedon 1999, 55, 11803-11818.
(51) Pudovik, A. N.; Konovalova, I. V. Synthesis 1979, 2, 81-96.
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5324 J . Org. Chem., Vol. 68, No. 13, 2003

Monofluoro LPA Analogues
Hama, Y. Xu, and G. D. Prestwich, in preparation).
Moreover, the R-monofluoromethylene-substituted LPA
analogue 3a a was 1000-fold more potent than natural
18:1 LPA for the LPA3 receptor.54 This response was
enantiospecific, indicating that the R-fluorophosphonates
are structurally informative and receptor-selective mim-
ics for phosphate in LPA. The full biological data and
enantioselectivity of the receptor recognition will be
reported in due course (J . Aoki, K. Hama, Y. Xu, and G.
D. Prestwich, in preparation).
Finally, the effects of monofluorinated sn-1 O-acyl LPA
analogues 2a -d were evaluated in a nuclear reporter
assay in which monocytic cells were transfected with a
luciferase construct activated by a PPARγ nuclear recep-
tor response element.5 As illustrated in Figure 3, ana-
logues 2a -d were essentially equipotent with sn-1-oleoyl-
LPA 4 in this assay. Importantly, compounds inactive
in the transmembrane receptor assays were fully active
as nuclear receptor ligands. These data demonstrate that
particular fluorine substitutions can give selective ago-
nists for different LPA receptors, and that some, but not
all, biological responses show both regioselectivity and
enantioselectivity relative to the placement of the acyloxy
and fluoro substituents.
In summary, we have described efficient methods for
the preparation of monofluoro-substituted and R-mono-
fluoromethylene phosphonate analogues of LPA from
readily available precursors. The intriguing biological
activity of LPA warrants a detailed evaluation of the
fluorinated structure-activity relationships of LPA ana-
logues for a variety of biological responses. Full descrip-
tions of biological activity and the utility of analogues as
migration-blocked or non-hydrolyzable LPA mimics will
be reported in due course.
F IGURE 2. Known synthetic ligands with LPA-like activities.
TABLE 1. Activa tion of P la telet Aggr ega tion for 1 µM
sn -1-O-Oleoyl LP A (1, R ) Oleoyl) a n d 1 µM of Ea ch of
F ou r LP A An a logu es 2a -d a
compd
aggregation response
1 (R ) oleoyl)
++
+
2a
2b
2c
2d
35
36
+
-
-
++
++
a
Key: ++, strong aggregation response; +, weak but pro-
nounced aggregation; -, no aggregation.
together with the 1,1-difluoro LPA derivatives35 (Figure
2, 35 and 36) and the R,R-difluoromethylene phospho-
nates45 described previously, now provide a set of ligands
to perform this comprehensive analysis.
Exp er im en ta l Section
Gen er a l P r oced u r es. Except where noted, all reagents
were purchased commercially. Solvents were of reagent grade
and were distilled before use: THF was dried by distillation
from sodium-benzophenone ketyl and methylene chloride was
distilled from CaH2. Reactions were performed under an inert
atmosphere (N2 or Ar) unless otherwise indicated. NMR
spectra were recorded at 400 (1H), 101 (13C), 162 (31P), and
376 MHz (19F), at 25 °C. Chemical shifts are reported relative
to those of internal chloroform (δH 7.24), methanol (δH 4.78),
or tetramethylsilane (δH 0.00) for 1H; chloroform (δC 77.0) or
methanol (δC 49.0) for 13C; and CFCl3 for 19F (δF 0.00); 85%
H3PO4 (δP 0.00) was used as an external standard. Optical
rotations were obtained at ambient temperature.
Three biological activities have been evaluated for
selected analogues: platelet activation, transmembrane
LPA receptor activation, and nuclear PPARγ activation.
Taken together, these assays demonstrate that different
analogues may activate quite different LPA-mediated
responses. First, platelet activation was evaluated by
using a standard aggregometer assay,53 in which acti-
vated platelets release additional LPA leading to platelet
aggregation1,6 (S. Smyth and A. Morris, personal com-
munication). As shown in Table 1, sn-1-O-oleoyl LPA (4)
and the two 1,1-difluoro analogues 35 and 36 (Figure 2)35
were potent activators of platelet aggregation at 1 µM.
In contrast, while the two (2S)-fluorodeoxy analogues 2a
and 2b showed weak platelet activation, the enantiomeric
(2R) analogues 2c and 2d were inactive.
Second, the LPA analogues were tested in insect cells
expressing LPA1, LPA2, or LPA3 receptors54 (J . Aoki, K.
Hama, Y. Xu, and G. D. Prestwich, in preparation). While
compounds 1a , 1b, and 2a -d failed to show either
significant agonist or antagonist activity for any of the
three isoforms, 1c was found to be more potent than
natural 18:1 LPA for the LPA3 receptor54 (J . Aoki, K.
Dim et h yl 1,2-(S)-Isop r op ylid en e-sn -glycer ol-3-p h os-
p h a te (6). t-BuOK (1.274 g, 11.35 mmol) was added to a
stirred solution of (R)-isopropylideneglycerol (1.00 g, 7.57
mmol) and dimethyl chlorophosphate (1.367 g, 9.46 mmol) in
CH2Cl2 (25 mL), and the solution was stirred at room tem-
perature for 1 h (complete by TLC). A saturated aq solution
of NH4Cl (40 mL) was added, the solution was stirred for 10
min, and the aq phase was extracted three times with CH2Cl2
(30 mL); the organic solution was dried (Na2SO4) and concen-
trated in vacuo. The crude product was purified on silica gel
by elution with diethyl ether to give 1.62 g (6.75 mmol, 92%
yield, Rf 0.30, diethyl ether) of pure product as a colorless oil.
δH(CDCl3): 4.22 (m, 1H), 3.95 (m, 4H), 3.69 (s, 3H), 3.66 (s,
3H), 1.33 (s, 3H), 1.24 (s, 3H). δH(CDCl3): 106.69 (s), 73.88 (d,
J ) 7.6 Hz), 67.36 (d, J ) 5.3 Hz), 65.84 (s), 54.23 (d, J ) 3.8
(53) Coller, B.; Peerschke, E.; Scudder, L.; Sullivan, C. J . Clin.
Invest. 1983, 72, 325-338.
(54) Prestwich, G. D.; Xu, Y.; Qian, L.; Hama, K.; Aoki, J . Annual
Meeting, ASBMB, San Diego, CA, 2003; poster 628.20.
Hz), 26.51 (s), 25.06 (s). δP(CDCl3): 2.23 (s). [R]20 +2.28° (c
D
2.08, MeOH).
J . Org. Chem, Vol. 68, No. 13, 2003 5325

Xu et al.
F IGURE 3. The PPARγ response element luciferase reporter stimulation in a cell-based assay (see Experimental Section) is
shown at 5 µM sn-1-O-oleoyl LPA (4, R ) oleoyl) and 5 µM of each of four LPA analogues 2a -d .
Dim eth yl (2S)-1,2-Di(tetr a bu tyld im eth ylsilyl)-sn -glyc-
er ol-3-p h osp h a te (8). TsOH (54 mg, 0.28 mmol, 0.10 eq) was
added to a solution of 6 (0.678 g, 2.83 mmol) in MeOH (10
mL), and the solution was stirred at room temperature for 24
h. After addition of NEt3 (0.1 mL), the solvent was removed
under reduced pressure. Following addition of anhydrous DMF
(3 mL), imidazole (0.577 g, 8.48 mmol, 3.0 equiv), and tert-
butyldimethylsilyl chloride (TBSCl) (1.107 g, 7.35 mmol, 2.8
equiv), the reaction mixture was stirred at room temperature
for an additional 36 h. The solution was diluted with water
(15 mL) and ethyl acetate (20 mL), and the aqueous layer was
separated and extracted three times with ethyl acetate (30
mL). The combined organic layers were dried (Na2SO4) and
concentrated in vacuo, and the residue was purified on silica
gel (n-hexane/ethyl acetate 4:1, Rf 0.13) to afford 0.804 g (1.88
mmol, 67%) of a colorless liquid. δH(CdCl3): 4.08 (m, 1H), 3.89
(m, 1H), 3.80 (m, 1H), 3.73 (d, J ) 1.2 Hz, 3H), 3.70 (d, J )
1.2 Hz, 3H), 3.51 (d, J ) 5.2 Hz, 3H), 0.84 (s, 9H), 0.84 (s,
9H), 0.04 (s, 3H), 0.03 (s, 3H), 0.01 (s, 3H), 0.00 (s, 3H).
δC(CdCl3): 84.77 (d, J ) 6.1 Hz), 77.50 (d, J ) 7.6 Hz), 74.36
(d, J ) 6.2 Hz), 69.50 (s), 67.52 (d, J ) 4.5 Hz), 59.69 (d, J )
6.3 Hz), 31.34 (s), 31.20 (s), 31.22 (s), 23.75 (s), 23.57 (s), 0.77
(s), 0.68 (s), 0.02 (s), 0.00 (s). δP(CdCl3): 2.42 (s). MS (CI) m/z
429.1 (M+ + 1, 100.00). HRMS for C17H42PSi2O6: found
mixture was added 0.2 mL of methanol followed by neutraliza-
tion with solid NaHCO3. After concentration in vacuo, the
residue was purified on silica gel (hexanes-ethyl acetate 1:1,
Rf 0.25) to afford 0.026 g. (0.083 mmol, 53%) as a colorless oil.
δH(CdCl3): 4.35 (ddd, 1H), 4.24 (ddd, 1H), 4.02-3.86 (m, 3H),
3.69 (d, J ) 1.2 Hz, 3H), 3.66 (d, J ) 1.2 Hz, 3H), 0.79 (s, 9H),
0.05 (s, 6H). δC(CdCl3): 88.46 (d, J ) 172.6 Hz), 74.76 (dd, J
) 20.7, 8.5 Hz), 72.26 (t, J ) 6.5 Hz), 59.31 (d, J ) 7.6 Hz),
30.55 (s), 22.98 (s), 0.00 (s). δP(CdCl3): 2.252 (s). δF(CdCl3):
230.50 (td, J ) 47.0, 20.7 Hz). MS (CI) m/z 317.1 (M+ + 1,
100.00). HRMS for C11H27FSiPO5: found 317.1344, calcd
317.1349. [R]20 +0.23° (c 0.33, MeOH).
D
1-P h osp h o-2(S)-(oleoyl)-3-flu or in e-p r op a n e-1,2-d iol Di-
m eth yl Ester (12a ). A solution of 10 (18 mg, 0.058 mmol) in
THF (2 mL) was treated consecutively with acetic acid (13 µL,
0.231 mmol) and tetrabutylammoniumfluoride trihydrate (73
mg, 0.231 mmol) at room temperature. After the solution was
stirred for 18 h the reaction was complete (TLC control), the
solvent was then evaporated under reduced pressure and the
crude product was purified on a short column of silica gel to
afford a colorless liquid. To the crude alcohol 11 and 42 mg
(47 µL, 0.147 mmol) of oleic acid in dry CH2Cl2 (1 mL) at room
temperature was added dropwise a solution of DCC (30 mg,
0.147 mmol) and DMAP (6.0 mg, 0.048 mmol) in dry CH2Cl2
(1 mL). The solution was stirred at room temperature for 18
h, filtered, concentrated in vacuo, and the residue was purified
on silica gel (n-hexanes-ethyl acetate 1:1, Rf 0.28) to afford
12 mg of a waxy solid (0.026 mmol, 45%). δH(CdCl3): 5.28 (m,
2H), 5.14 (dm, J ) 20.8 Hz, 1H), 4.51 (dd, J ) 46.8, 4.0 Hz,
2H), 4.15 (m, 2H), 3.73 (d, J ) 2.4 Hz, 3H), 3.70 (d, J ) 2.4
Hz, 3H), 2.30 (t, J ) 7.2 Hz, 2H), 1.90 (m, 4H), 1.56 (m, 4H),
1.14 (m, 20H), 0.81 (t, J ) 6.4 Hz, 3H). δC(CdCl3): 173.00 (s),
130.26 (s), 129. 93 (s), 80.22 (d, J ) 172.0 Hz), 70.29 (d, J )
28.6 Hz), 64.64 (t, J ) 6.5 Hz), 54.74 (s), 54.68 (s), 34.32 (s),
34.17 (s), 32.12 (s), 29.98 (s), 29.90 (s), 29.53 (s), 29.36 (s), 29.30
(s), 29.24 (s), 27.44 (s), 27.38 (s), 25.84 (s), 25.16 (s), 25.01 (s),
22.89 (s), 14.32 (s). δP(CdCl3): 2.185 (s). δF(CdCl3): -234.50
(td, J ) 47.0, 20.7 Hz). MS (CI) m/z 467.0 (M+ + 1, 100.00),
341.2 (M+ - OPO(OMe)2, 56.20). HRMS for C23H45FPO6: found
429.2244, calcd 429.2230. [R]20 +0.18° (c 2.25, MeOH).
D
Dim eth yl (2S)-(Tetr a bu tyld im eth ylsilyl)-sn -glycer ol-
3-p h osp h a te (9). The HF‚pyridine complex (70%, 0.31 mL)
was added to a mixture of pyridine (1.40 mL) and a solution
of the bis-TBS ether 8 (0.759 g, 1.77 mmol) in THF (10 mL).
The reaction mixture was stirred for 24 h. After completion of
the reaction (TLC), the solution was diluted with ethyl acetate
(50 mL), washed with saturated NaCl solution (5 mL), and
dried over anhydrous Na2SO4. After removal of the solvents,
the residue was purified on silica gel (ethyl acetate, Rf 0.23)
to afford a colorless liquid: 0.254 g (0.814 mmol, 46%).
δH(CdCl3): 3.93 (m, 2H), 3.82 (m, 1H), 3.69 (d, J ) 1.2 Hz),
3.66 (d, J ) 1.2 Hz, 3H), 3.52 (dd, J ) 8.4, 4.4 Hz, 2H), 0.79
(s, 9H), 0.01 (s, 3H), 0.00 (s, 3H). δC(CdCl3): 76.06 (d, J ) 7.6
Hz), 72.40 (d, J ) 6.1 Hz), 67.93 (s), 59.29 (d, J ) 6.1 Hz),
30.57 (s), 22.91 (s), 0.11 (s), 0.00 (s). δP(CdCl3): 2.788 (s). MS
(CI) m/z 315.1 (M+ + 1, 100.00). HRMS for C11H28SiPO6: found
467.2921, calcd 467.2904. [R]20 +0.69° (c 0.36, MeOH).
D
1-P h osp h o-2(S)-(p a lm itoyl)-3-flu or in e-p r op a n e-1,2-d i-
ol Dim eth yl Ester (12b). A solution of 10 (22 mg, 0.071
mmol) in THF (2 mL) was treated consecutively with acetic
acid (16 µL, 0.28 mmol) and tetrabutylammoniumfluoride
trihydrate (89 mg, 0.28 mmol) at room temperature. The crude
alcohol 11 was directly esterified with palmitic acid (following
the protocol above for 12a ) and purified on silica gel (n-
hexanes-ethyl acetate 1:1, Rf 0.28) to afford 11 mg of a waxy
315.1412, calcd 315.1414. [R]20 +0.28° (c )1.08, MeOH).
D
1-P h osp h o-2(S)-(t et r a b u t yld im et h ylsilyl)-3-flu or in e-
p r op a n e-1,2-d iol Dim eth yl Ester (10). To a mixture of 0.035
g (0.220 mmol) of DAST and 2 mL of dry CH2Cl2 at -78 °C
was added dropwise a solution of alcohol (0.049 g, 0.157 mmol)
in 1 mL of dry CH2Cl2. The mixture was stirred at -78 °C for
1 h, then at room temperature for an additional 1 h. To the
5326 J . Org. Chem., Vol. 68, No. 13, 2003

Monofluoro LPA Analogues
solid (0.025 mmol, 35%). δH(CdCl3): 5.20 (dm, J ) 21.0 Hz,
1H), 4.57 (dd, J ) 46.8, 4.0 Hz, 2H), 4.25 (m, 2H), 3.79 (d, J )
2.8 Hz, 3H), 3.76 (d, J ) 2.4 Hz, 3H), 2.36 (t, J ) 9.6 Hz, 2H),
1.93 (m, 2H), 1.62 (m, 4H), 1.24 (m, 20H), 0.87 (t, J ) 9.6 Hz,
3H). δC(CdCl3): 173.0 (s), 80.84 (d, J ) 173.4 Hz), 70.27 (d, J
) 7.64 Hz), 70.07 (d, J ) 7.4 Hz), 64.64 (t, J ) 6.7 Hz), 54.74
(s), 54.68 (s), 29.88-29.86 (m), 29.81 (s), 29.57 (s), 29.45 (s),
29.27 (s). δP(CdCl3): 2.171 (s). δF(CdCl3): -234.49 (td, J ) 47.0,
21.0 Hz). MS (CI) m/z 441.3 (M+ + 1, 20.84), 225 (M+ - H2O
- C12H25, 100.00). HRMS for C21H43FPO6: found 441.2790,
0.81 (t, J ) 6.4 Hz, 3H). δC(Cd3OD): 173.80 (s), 130.86 (s),
130.53 (s), 80.72 (d, J ) 171.9 Hz), 70.79 (d, J ) 28.4 Hz),
65.09 (t, J ) 6.5 Hz), 34.75 (s), 34.60 (s), 33.72 (s), 33.55 (s),
31.87 (s), 29.65 (s), 29.60 (s), 29.41 (s), 29.25 (s), 29.15 (s), 29.08
(s), 28.98 (s), 28.91 (s), 26.93 (s), 14.35 (s). δP(Cd3OD): 0.840
(s). δF(Cd3OD): -235.96 (td, J ) 46.6, 20.6 Hz). [R]20 -0.71°
D
(c 0.29, MeOH). The compound was converted to the sodium
salt by ion exchange and stored in solid form at -80 °C as
described for 1a .
Dim eth yl 1-(Tetr abu tyldiph en ylsilyl)-2-(R)-sn -glycer ol-
3-p h osp h a te (15). TsOH (0.594 g, 3.0 mmol, 0.15 equiv) was
added to a solution of dimethyl 1,2-(R)-isopropylidene-sn-
glycerol-3-phosphate (4.80 g, 20.00 mmol) in MeOH (100 mL),
and the solution was stirred at room temperature for 24 h.
Following addition of solid NaHCO3, the mixture was filtered,
concentrated in vacuo, and purified on silica gel (methanol:
ethyl acetate 1:5, Rf 0.26) to afford 3.64 g (18.2 mmol, 91%) of
diol 14 as a colorless liquid. To a solution of the crude diol 14
(3.45 g, 17.25 mmol) in anhydrous DMF (120 mL) was added
imidazole (3.41 g, 50.0 mmol, 2.9 equiv) and TBDPS chloride
(6.16 g, 22.4 mmol, 1.3 equiv). The reaction mixture was stirred
at 0 °C for 8 h, then at room temperature for 12 h. The solution
was diluted with ethyl acetate (100 mL), and the solution was
washed with saturated aq NH4Cl solution and brine. After
drying with anhydrous Na2SO4, the organic layer was concen-
trated in vacuo and purified on silica gel (ethyl acetate, Rf 0.48)
to afford 5.10 g of a colorless liquid (11.68 mmol, 68%).
δH(CDCl3): 7.65 (m, 4H), 7.36 (m, 6H), 4.16 (m, 2H), 3.93 (m,
1H), 3.71 (d, J ) 3.0 Hz, 3H), 3.68 (d, J ) 2.0 Hz, 3H), 1.04 (s,
9H). δC(CDCl3): 135.20 (s), 135.18 (s), 132.74 (s), 132.73 (s),
129.51 (s), 127.47 (s), 70.20 (d, J ) 6.1 Hz), 68.52 (d, J ) 6.1
Hz), 63.61 (s), 54.05 (dd, J ) 6.1, 2.3 Hz), 26.49 (s), 18.88 (s).
δP(CDCl3): 2.869 (s). MS (CI) m/z 438.9 (M+ + 1, 20.62), 380.9
(M+ - C4H9, 39.84), 360.9 (M+ - C6H5, 100.00). HRMS for
calcd 441.2781. [R]20 +0.91° (c 0.29, MeOH).
D
1-P h osph o-2(S)-(oleoyl)-3-flu or in e-pr opan e-1,2-diol (1a).
A thoroughly dried sample of ester 12a (8 mg, 0.017 mmol, 5
h under high vacuum) was dissolved in dry methylene chloride
(1 mL) at room temperature. Next, bromotrimethylsilane
(TMSBr, 9 µL, 0.052 mmol) was added via syringe and the
reaction was stirred for 4 h. When TLC indicated that all of
the reactant had been consumed, the solvent was removed
under reduced pressure and the residue was dried in vacuo.
The residue was dissolved in 95% methanol (1 mL) for 1 h,
the solvent was then removed under reduced pressure, and
the product was dried in vacuo to give 6 mg of a colorless oil
(CH2Cl2:CH3OH:H2O 20:10:1, Rf 0.39, 0.014 mmol, 82% yield).
Without further purification, the purity of this product 1a was
>98% homogeneous as determined by TLC and NMR (1H, 13C,
19F, and 31P). δH(Cd3OD): 5.24 (m, 2H), 5.11 (dm, J ) 20.4
Hz, 1H), 4.49 (dd, J ) 47.2, 4.8 Hz, 2H), 4.03 (m, 2H), 2.29 (t,
J ) 7.6 Hz, 2H), 1.93 (m, 4H), 1.61-1.54 (m, 4H), 1.20 (m,
17H), 0.81 (t, J ) 6.4 Hz, 3H). δC(Cd3OD): 173.80 (s), 130.86
(s), 130.53 (s), 80.72 (d, J ) 171.9 Hz), 70.79 (d, J ) 28.4 Hz),
65.09 (t, J ) 6.5 Hz), 34.75 (s), 34.60 (s), 33.72 (s), 33.55 (s),
31.87 (s), 29.65 (s), 29.60 (s), 29.41 (s), 29.25 (s), 29.15 (s), 29.08
(s), 28.98 (s), 28.91 (s), 26.93 (s), 14.35 (s). δP(Cd3OD): 0.843
(s). δF(Cd3OD): -235.96 (td, J ) 47.0, 20.7 Hz). m/z 438.0 (M+,
0.30), 314.2, (M+ - OPO(OH)2, 100.00), 157, (M+ - OCOR,
C
21H32O6PSi (M+ + 1): found 439.1685, calcd 439.1706. [R]20
D
62.91). MS (CI) m/z 439.3 (M+ + 1, 45.34). HRMS for C21H41
-
-0.77 (c 0.31, MeOH).
FO6P (M+ + 1): found 439.2634, calcd 439.2625. [R]20D +0.57°
(c 0.12, MeOH).
1-P h osp h o-2(S)-flu or in e-3-(t et r a b u t yld ip h en ylsilyl)-
p r op a n e-1,3-d iol Dim eth yl Ester (16). To a mixture of
DAST (1.77 g, 10.96 mmol) and 50 mL of dry CH2Cl2 at -78
°C was added dropwise a solution of alcohol (4.00 g, 9.13 mmol)
in 20 mL of dry CH2Cl2. The mixture was stirred at -78 °C
for 1 h, followed by 1 h at room temperature. The mixture was
poured into a stirred mixture of saturated NaHCO3 and ice
chips, then extracted with CH2Cl2. The extract was washed
with H2O, dried (Na2SO4), filtered, and evaporated under
reduced pressure. The oil was purified on silica gel (hexanes:
ethyl acetate 1:1, Rf 0.19) to afford 1.53 g (3.47 mmol, 38%) of
16 as a colorless liquid. δH(CDCl3): 7.64 (m, 4H), 7.42 (m, 6H),
4.71 (dm, J ) 47.6 Hz, 1H), 4.30 (dm, J ) 23.6 Hz, 2H), 3.83
(m, 2H), 3.76 (d, J ) 2.4 Hz, 3H), 3.68 (d, J ) 2.4 Hz, 3H),
1.04 (s, 9H). δC(CDCl3): 135.55 (s), 135.49 (s), 132.79 (s), 132.67
(s), 129.90 (s), 127.81 (s), 127.79 (s), 91.17 (dd, J ) 177.2, 6.9
Hz), 66.33 (dd, J ) 23.7, 5.3 Hz), 62.27 (d, J ) 25.3 Hz), 54.40
(d, J ) 6.1 Hz), 26.68 (s), 19.19 (s). δF(CDCl3): -196.16 (1F,
m). δP(CDCl3): 2.278 (s). MS (CI) m/z 383.0 (M+ - C4H9, 29.86),
The labile acid forms of these analogues were then converted
to mixed neutral sodium salts. Thus, product 1a was dissolved
in 2 mL of 1.0 M triethylammonium bicarbonate (TEAB) buffer
(pH 8.0) to give a slightly cloudy solution, which was absorbed
to a sodium ion-exchange column (Dowex 50WX8-200 resin,
neutral Na+ form). The desired mixed neutral sodium salt of
1a was eluted with Nanopure water. The product solution was
lyophilized to give sodium salt as white amorphous solid, which
was stored in solid form at -80 °C. Aqueous solutions of LPA
analogues were prepared and used within several days to
minimize hydrolysis and acyl migration (when possible). All
LPA analogues described herein were converted to the corre-
sponding sodium salts in the same procedure.
1-P h osp h o-2(S)-(p a lm itoyl)-3-flu or in e-p r op a n e-1,2-d i-
ol (1b). Deprotection of 12b (11 mg, 0.025 mmol, 5 h drying
at 0.01 mg Hg) was conducted as described above for 12a to
give 6 mg of phosphate 1b as a colorless oil (CH2Cl2:CH3OH:
H2O 20:10:1, Rf 0.37, 0.019 mmol, 78% yield). δH(Cd3OD): 5.22
(dm, J ) 21.0 Hz, 1H), 4.58 (dd, J ) 47.2, 3.2 Hz, 2H), 4.25
(m, 2H), 2.36 (t, J ) 9.6 Hz, 2H), 1.93 (m, 2H), 1.76 (m, 2H),
1.62 (m, 4H), 1.29 (m, 18H), 0.87 (t, J ) 6.8 Hz, 3H).
δC(Cd3OD): 173.40 (s), 81.24 (d, J ) 173.3 Hz), 70.67 (d, J )
7.5 Hz), 70.47 (d, J ) 7.4 Hz), 64.95 (t, J ) 6.6 Hz), 32.78 (s),
32.14 (s), 29.93 (s), 29.88 (s), 29.71 (s), 29.59 (s), 25.26 (s), 25.00
(s), 24.63 (s), 22.91 (s), 14.32 (s). δP(Cd3OD): 1.742 (s).
δF(Cd3OD): -234.63 (td, J ) 46.0, 21.0 Hz). MS (CI) m/z 413.3
(M+ + 1, 51.22). HRMS for C19H39FO6P (M+ + 1): found
363.0 (M+ - C6H5, 100.00). HRMS for C17H21FO5PSi (M+
-
C4H9): found 383.0875, calcd 383.0880. [R]20 -4.88° (c 0.42,
D
MeOH).
1-P h osp h o-2(S)-flu or in e-pr opa n e-1,3-diol Dim eth yl Es-
ter (17). A solution of 16 (860 mg, 1.972 mmol) in THF (50
mL) was treated consecutively with acetic acid (0.46 mL, 7.888
mmol) and tetrabutylammoniumfluoride trihydrate (2.489 g,
7.888 mmol) at room temperature. After the solution was
stirred for 16 h the reaction was complete (TLC), and the
mixture was concentrated and passed through a silica column
(ethyl acetate, Rf 0.20) to afford 0.342 g (1.69 mmol, 86%) of
17 as a colorless liquid. δH(CDCl3): 4.67 (dm, J ) 48.0 Hz,
1H), 4.23 (ddd, J ) 22.4, 7.6, 4.4 Hz, 2H), 3.77 (dm, J ) 19.6
Hz, 2H), 3.75 (d, J ) 2.0 Hz, 3H), 3.72 (d, J ) 2.0 Hz, 3H),
3.48 (br, 1H). δC(CDCl3): 91.32 (dd, J ) 174.8, 6.1 Hz), 66.02
(dd, J ) 23.7, 5.3 Hz), 60.53 (d, J ) 23.8 Hz), 54.54 (dd, J )
6.1, 3.8 Hz). δF(CDCl3): -197.66 (1F, m). δP(CDCl3): 2.453 (s).
413.2479, calcd 413.2468 [R]20 +0.81° (c 0.14, MeOH). The
D
compound was converted to the sodium salt by ion exchange
and stored in solid form at -80 °C as described for 1a .
1-P h osph o-2(S)-(oleoyl)-3-flu or in e-pr opan e-1,2-diol (1c).
Colorless oil, δH(Cd3OD): 5.24 (m, 2H), 5.11 (dm, J ) 20.4 Hz,
1H), 4.49 (dd, J ) 47.2, 4.8 Hz, 2H), 4.03 (m, 2H), 2.29 (t, J )
7.6 Hz, 2H), 1.93 (m, 4H), 1.61-1.54 (m, 4H), 1.20 (m, 17H),
J . Org. Chem, Vol. 68, No. 13, 2003 5327

Xu et al.
MS (CI) m/z 203.1 (M+ + 1, 100.00). HRMS for C5H12FO5P (M+
+ 1): found 203.0476, calcd 203.0485.
salt by ion exchange and stored in solid form at -80 °C as
described for 1a .
1-P h osp h o-2(S)-flu or in e-3-(oleoyl)-p r op a n e-1,3-d iol
Dim eth yl Ester (18a ). To a solution of crude alcohol 17 (73
mg, 0.361 mmol) with oleic acid (113 mg, 0.397 mmol) in dry
CH2Cl2 (3 mL) at room temperature was added dropwise a
solution of DCC (112 mg, 0.542 mmol) and DMAP (27 mg,
0.217 mmol) in dry CH2Cl2 (3 mL). The solution was stirred
at room temperature for 16 h and filtered, the solvent was
removed, and the residue was purified on silica gel (n-hexanes:
ethyl acetate 1:2, Rf 0.30) to afford 162 mg (0.347 mmol, 96%)
of 18a as a waxy solid. δH(CDCl3): 5.28 (m, 2H), 4.80 (dm, J
) 47.6 Hz, 1H), 4.24 (m, 4H), 3.74 (s, 3H), 3.72 (s, 3H), 2.86
(t, J ) 7.2 Hz), 1.94 (m, 4H), 1.56 (m, 2H), 1.22 (m, 20H), 0.81
(t, J ) 8.0 Hz, 3H). δC(CDCl3): 173.07 (s), 129.87 (s), 129.57
(s), 88.67 (dd, J ) 178.0, 7.6 Hz), 65.77 (dd, J ) 24.5, 5.3 Hz),
61.97 (d, J ) 23.7 Hz), 54.39 (d, J ) 6.1 Hz), 33.80 (s), 31.77
(s), 29.63 (s), 29.54 (s), 29.38 (s), 29.18 (s), 29.00 (s), 28.94 (s),
28.92 (s), 27.07 (s), 27.02 (s), 24.67 (s), 22.54 (s), 13.96 (s).
δF(CDCl3): -195.98 (1F, m). δP(CDCl3): 2.151 (s). MS (CI) m/z
467.4 (M+ + 1, 100.00), 341.3 (M+ - C2H6PO4, 32.11). HRMS
for C23H45FO6P (M+ + 1): found 467.2891, calcd 467.2938.
1-P h osp h o-2(R)-flu or in e-3-p a lm it oyl-p r op a n e-1,3-d i-
ol (2d ) was obtained similarly as a white solid. [R]20 -4.51°
D
(c 0.24, MeOH/H2O 1/1, v/v). The compound was converted to
the sodium salt by ion exchange and stored in solid form at
-80 °C as described for 1a .
Dieth yl [1-F lu or o-3,4-ep oxy-bu tyl]p h osp h on a te (22).
K2CO3 (0.375 g, 2.71 mmol) was added to a solution of
iodohydrin 21 (0.160 g, 0.452 mmol) in MeOH (20 mL). The
reaction mixture was stirred for 10 min at room temperature,
diluted with water, and extracted with CH2Cl2. The organic
phase was dried (Na2SO4), filtered, and concentrated in vacuo.
The residue was purified by flash chromatography on silica
gel to give 69 mg. (0.307 mmol, 68%, n-hexanes:ethyl acetate
1:2, Rf 0.21) of epoxide 22 as a colorless liquid. δH(CDCl3):
4.94-4.70 (m, 1H), 4.18-4.09 (m, 4H), 3.09 (m, 1H), 2.79 (t, J
) 4.8 Hz, 0.5H), 2.72 (t, J ) 4.4 Hz, 0.5H), 2.50 (m, 1H), 2.21-
2.08 (m, 2H), 1.28 (m, 6H). δC(CDCl3): 86.85 (dd, J ) 172. 6,
148.0 Hz), 86.32 (dd, J ) 172. 6, 148.0 Hz), 63.24 (dd, J ) 7.6,
3.8 Hz), 62.88 (dd, J ) 10.8, 6.1 Hz), 48.40 (dd, J ) 14.6, 3.8
Hz), 48.17 (dd, J ) 16. 9, 3.8 Hz), 47.54 (s), 46.32 (s), 33.73
(dd, J ) 20.6, 1.5 Hz), 32.79 (dd, J ) 19.9, 1.5 Hz), 16.33 (d, J
) 3.0 Hz), 16.27 (d, J ) 3.1 Hz). δF(CDCl3): -207.82 (0.5F,
m), -211.22 (0.5F, m). δP(CDCl3): 18.02 (0.5d, J ) 73.8 Hz),
17.97 (0.5d, J ) 75.0 Hz). MS (CI) m/z 227.1 (M+ + 1, 15.81),
203.1 (M+ + 1, 11.28). HRMS for C8H17FO4P (M+ + 1): found
227.0836, calcd 227.0849.
Hyd r olytic Kin etic Resolu tion of Ep oxid e 22. A 10-mL
flask equipped with a stir bar was charged with (R,R)-23 (26.7
mg, 43 µmol, 0.01 equiv). The catalyst was dissolved in 0.4
mL of PhMe and treated with AcOH (10 µL, 0.177 mmol). The
solution was allowed to stir at room temperature open to air
for 30 min; the color changed from orange-red to a dark brown.
The solution was concentrated in vacuo to leave a crude brown
solid. The resulting catalyst residue was dissolved in a solution
of epoxide 22 (1.00 g, 4.425 mmol) and THF (150 µL) at room
temperature, the reaction flask was cooled to 0 °C, and H2O
(36 µL, 1.991 mmol, 0.45 equiv) was added dropwise over 5
min. The reaction was allowed to warm to room temperature
with stirring for 14 h. The reaction mixture was diluted with
20 mL of CH2Cl2 and the precipitate was removed by passage
through Celite 351. Flash chromatography on silica gel af-
forded (R)-epoxide 25a (0.485 g, 2.146 mmol, 97%, Rf 0.32,
CH2Cl2:CH3OH 20:1) and (S)-diol 24a (0.394 g, 1.615 mmol,
73%, Rf 0.34, CH2Cl2:CH3OH 10:1). The ee value of 24a was
91%, which is obtained by conversion to the known25 isopro-
pylidene-protected ketal. A comparison of the reported optical
rotation values was then made.
[R]20 -1.92° (c 2.52, MeOH).
D
1-P h osp h o-2(S)-flu or in e-3-(p a lm itoyl)-p r op a n e-1,3-d i-
ol Dim eth yl Ester (18b). The same procedure was followed
as for 18a to give 18b as a waxy solid (n-hexanes:ethyl acetate
1:2, Rf 0.30; 139 mg, 0.316 mmol, 91%). δH(CD3Cl): 4.77 (dm,
J ) 48.0 Hz, 1H), 4.17 (m, 4H), 3.77 (s, 3H), 3.68 (s, 3H), 2.26
(t, J ) 7.6 Hz, 2H), 1.53 (m, 2H), 1.16 (m, 24H), 0.78 (t, J )
6.4 Hz, 3H). δC(CD3OD): 173.43 (s), 88.57 (dd, J ) 178.7, 7.6
Hz), 65.87 (dd, J ) 23.8, 5.4 Hz), 61.92 (d, J ) 23.8 Hz), 54.43
(d, J ) 6.1 Hz), 33.77 (s), 31.72 (s), 29.49 (s), 29.45 (s), 29.39
(s), 29.25 (s), 29.16 (s), 29.03 (s), 28.89 (s), 24.62 (s), 22.48 (s),
13.87 (s). δF(CD3OD): -196.11 (1F, m). δP(CD3OD): 1.977 (s).
MS (CI) m/z 441.3 (M+ + 1, 100.00), 315.3 (M+ - C2H6PO4,
38.53). HRMS for C21H43FO6P (M+ + 1): found 441.2770, calcd
441.2781. [R]20 -1.25° (c 1.25, CHCl3).
D
1-P h osp h o-2(S)-flu or in e-3-oleoyl-p r op a n e-1,3-d iol (2a ).
Following the same procedure used above for 1a afforded
analogue 2a as a white solid in 86% yield. δH(CD3OD/CDCl3,
2/1): 5.32 (m, 2H), 4.82 (dm, J ) 48.0 Hz, 1H), 4.37 (m, 2H),
4.05 (ddd, J ) 48.0, 5.8, 5.2 Hz, 2H), 2.35 (t, J ) 7.6 Hz, 3H),
2.00 (m, 4H), 1.62 (m, 2H), 1.29 (m, 20H), 0.87 (t, J ) 6.4 Hz,
3H). δC(CD3OD/CDCl3, 2/1): 174.10 (s), 129.86 (s), 129.69 (s),
90.70 (dd, J ) 175.0, 7.6 Hz), 64.47 (dd, J ) 24.5, 5.4 Hz),
64.13 (d, J ) 22.2 Hz), 34.63 (s), 32.64 (s), 30.45 (s), 30.40 (s),
30.22 (s), 30.03 (s), 29.97 (s), 29.89 (s), 29.79 (s), 27.82 (s), 27.80
(s), 25.57 (s), 23.35 (s), 14.37 (s). δF(CD3OD/CDCl3, 2/1):
-196.35 (1F, m). δP(CD3OD/CDCl3, 2/1): 2.145 (s). MS (CI)
m/z 437.2 (M+ + 1 - 2Na+, 86.37). HRMS for C21H39FO6P (M+
+ 1 - 2Na+): found 437.2429, calcd 437.2390. [R]20D +0.57° (c
0.58, MeOH). The compound was converted to the sodium salt
by ion exchange and stored in solid form at -80 °C as described
for 1a .
Dieth yl [1-F lu or o-3(S)-4-d ih yd r oxybu tyl]p h osp h on a te
(24a ) was obtained as described above as a colorless liquid.
δH(CDCl3): 5.13-4.88 (m, 1H), 4.21-4.05 (m, 4H), 3.97-3.85
(br, 2H), 3.61-3.41 (m, 3H), 2.12-1.94 (m, 2H), 1.31 (m, 6H).
δC(CDCl3): 86.16 (dd, J ) 171.0, 180.0 Hz), 85.54 (dd, J )
171.0, 180.0 Hz), 68.34 (dd, J ) 9.3, 3.1 Hz), 67.23 (dd, J )
14.2, 1.8 Hz), 66.59 (s), 65.88 (s), 63.65 (d, J ) 7.6 Hz), 63.44
(d, J ) 6.8 Hz), 63.19 (d, J ) 6.9 Hz), 63.12 (d, J ) 6.1 Hz),
33.87 (d, J ) 20.0 Hz), 33.68 (d, J ) 19.1 Hz), 16.34 (d, J )
5.3 Hz), 16.29 (d, J ) 4.6 Hz). δF(CDCl3): -207.48 (0.5F, m),
-211.53 (0.5F, m). δP(CDCl3): 19.91 (0.5P, d, J ) 75.0 Hz),
19.40 (0.5P, d, J ) 76.1 Hz). MS (CI) m/z 245.2 (M+ + 1,
100.00), 231.1 (M+ + 2 - CH3, 3.27). HRMS for C8H19FO5P
1-P h osp h o-2(S)-flu or in e-3-p a lm it oyl-p r op a n e-1,3-d i-
ol (2b) was obtained similarly as above as a white solid in
91% yield. δH(D2O/CD3OD): 4.81 (dm, J ) 48.8 Hz, 1H), 4.24
(dd, J ) 7.6, 6.4 Hz, 2H), 3.87 (dm, J ) 5.7 Hz, 2H), 2.27 (t, J
) 5.2 Hz, 2H), 1.49 (m, 2H), 1.16 (m, 24H), 0.76 (t, J ) 6.0
Hz, 3H). δC(D2O/CD3OD): 173.43 (s), 88.57 (dd, J ) 178.7, 7.6
Hz), 65.87 (dd, J ) 23.8, 5.4 Hz), 61.92 (d, J ) 23.8 Hz), 33.77
(s), 31.72 (s), 29.49 (s), 29.45 (s), 29.39 (s), 29.25 (s), 29.16 (s),
29.03 (s), 28.89 (s), 24.62 (s), 22.48 (s), 13.87 (s). δF(D2O/CD3OD):
-194.87 (1F, m). δP(D2O/CD3OD): 4.325 (s). MS (CI) m/z 441.4
(M+ + 1 - 2Na+, 100.00). HRMS for C19H43FO6P (M+ + 1):
(M+ + 1): found 245.0965, calcd 245.0954. [R]20 -18.77 (c
D
3.08, MeOH).
Dieth yl [1-Diflu or o-3(R)-3,4-ep oxy-bu tyl]p h osp h on a te
(25a ). 25a was recovered in resolved form as described above
as a colorless liquid. δC(CDCl3): 4.97-4.72 (m, 1H), 4.21-4.12
(m, 4H), 3.14-3.10 (m, 1H), 2.83 (t, J ) 4.0 Hz, 0.5H), 2.75 (t,
J ) 4.0 Hz, 0.5H), 2.54 (m, 1H), 2.29-2.08 (m, 2H), 1.32 (m,
6H). δC(CDCl3): 85.92 (dd, J ) 180.9, 172.5 Hz), 86.17 (dd, J
) 180.2, 172. 6 Hz), 63.35 (d, J ) 3.1 Hz), 63.28 (d, J ) 3.1
Hz), 63.00 (d, J ) 4.6 Hz), 62.93 (d, J ) 4.6 Hz), 48.49 (dd, J
found 411.2307, calcd 411.2312. [R]20 -5.00° (c 0.08, MeOH/
D
H2O, 1/1, v/v). The compound was converted to the sodium salt
by ion exchange and stored in solid form at -80 °C as described
for 1a .
1-P h osp h o-2(R)-flu or in e-3-oleoyl-p r op a n e-1,3-d iol (2c)
was obtained similarly as above as a white solid. [R]20D -0.69°
(c 0.45, MeOH). The compound was converted to the sodium
5328 J . Org. Chem., Vol. 68, No. 13, 2003

Monofluoro LPA Analogues
) 14.6, 3.8 Hz), 48.26 (dd, J ) 17.6, 3.8 Hz), 47.63 (s), 46.41
(s), 37.80 (d, J ) 19.8 Hz), 32.85 (d, J ) 19.9 Hz), 16.40 (d, J
) 12.4 Hz), 16.35 (d, J ) 12.0 Hz). δF(CDCl3): -207.73 (0.5F,
FO6P (M+ + 1): found 509.3400, calcd 509.3407. [R]20 -2.61
D
(c 2.38, MeOH).
Dieth yl[1-flu or o-3(S)-h yd r oxyl-4-(p a lm itoyloxy)bu tyl]-
p h osp h on a te (26a b) was obtained similarly as above as a
white solid, 51% yield. δH(CDCl3): 5.11-4.90 (m, 1H), 4.23-
3.99 (m, 7H), 3.42 (br, 1H), 2.31 (t, J ) 7.6 Hz, 2H), 2.19-1.90
(m, 2H), 1.68-1.55 (m, 2H), 1.33 (t, J ) 6.8 Hz, 6H), 1.60
(m, 24H), 0.84 (t, J ) 7.2 Hz, 3H). δC(CDCl3): 173.92 (s),
173.89 (s), 86.56 (dd, J ) 171.0, 168.2 Hz), 84.78 (dd, J ) 171.0,
168.2 Hz), 68.10 (s), 67.53 (s), 66.11 (dd, J ) 9.3, 3.8 Hz),
65.21 (dd, J ) 13.0, 3.1 Hz), 63.48 (dd, J ) 24.6, 6.9 Hz), 63.05
(dd, J ) 9.3, 6.8 Hz), 49.03 (s), 34.36 (d, J ) 19.9 Hz), 31.87
(s), 29.63 (s), 29.60 (s), 29.41 (s), 29.22 (s), 29.09 (s), 25.59 (s),
24.86 (s), 22.63 (s), 16.41 (d, J ) 5.3 Hz), 16.37 (d, J ) 4.6
Hz), 14.06 (s). δF(CDCl3): -208.37 (0.5F, m), -211.62 (0.5F,
m). δP(CDCl3): 19.34 (0.5P, d, J ) 73.8 Hz), 19.11 (0.5P, d,
m), -211.17 (0.5F, m). δP(CDCl3): 18.07 (d, J ) 73.8 Hz). [R]20
D
+9.75 (c 3.54, MeOH).
To obtain the enantiomeric diol 24b, the enantiomeric
catalyst was employed as follows. A 10-mL flask equipped with
a stir bar was charged with (S,S)-23 (20.3 mg, 34 µmol, 0.01
equiv). The catalyst was dissolved in 0.4 mL of PhMe and
treated with AcOH (7 µL, 0.134 mmol). The solution was
allowed to stir at room temperature open to air for 30 min;
the color changed from orange-red to a dark brown. The
solution was concentrated in vacuo to leave a crude brown
solid. The resulting catalyst residue was dissolved in epoxide
(0.758 g, 3.35 mmol) and THF (120 µL) at room temperature,
the reaction flask was cooled to 0 °C, and H2O (27 µL, 1.51
mmol, 0.45 equiv) was added dropwise over 5 min. The reaction
was allowed to warm to room temperature, stirred for 14 h,
concentrated, and purified on silica gel to give (S)-epoxide 25b
(0.369 g, 1.63 mmol, 98%) and (S)-diol 24b (0.375 g, 1.54 mmol,
90%). The ee value of diol 24b was 89%, obtained by conversion
of 24b to the known25 ketal and comparison of the reported
optical rotations.
J ) 76.1 Hz). MS (CI) m/z 483.4 (M+ + 1, 55.29), 437.4 (M+
-
OC2H5, 100.00). HRMS for C24H49FO6P (M+ + 1): found
483.3244, calcd 483.3251. [R]20 -2.20 (c 1.00, MeOH).
D
[1-F lu or o-3(S)-h yd r oxyl-4-(oleoyloxy)bu t yl]p h osp h o-
n a te (3a a ). Thoroughly dried precursor 26a a (117 mg, 0.203
mmol, 5 h under high vacuum) was dissolved in dry methylene
chloride (1 mL) at room temperature, bromotrimethylsilane
(353 mg, 2.03 mmol) was added with a dry syringe, and the
mixture was stirred for 4 h. When TLC indicated that all of
the reactant had been consumed, the solvents were removed
in vacuo. The residue was dissolved in 95% methanol (1 mL)
for 1 h and reconcentrated in vacuo to give a final product (88
mg, 0.195 mmol, 96% yield) of phosphonate 3a a . δH(CD3OD):
5.34 (m, 2H), 5.21-5.17 (m, 1H), 4.79 (m, 1H), 3.68 (dd, J )
11.60, 4.40 Hz, 1H), 3.57 (m, 1H), 2.35 (m, 4H), 2.01 (m, 4H),
1.63 (m, 2H), 1.33-1.22 (m, 20H), 0.89 (t, J ) 7.2 Hz, 3H).
δC(CDCl3): 174.33 (s), 174.17 (s), 130.84 (s), 130.74 (s), 88.16
(dd, J ) 170.3, 168.7 Hz), 86.39 (dd, J ) 170.3, 168.7 Hz), 71.30
(dd, J ) 14.6, 2.3 Hz), 69.52 (dd, J ) 14.6, 2.3 Hz), 35.12 (d, J
) 19.3 Hz), 34.93 (d, J ) 18.9 Hz), 33.04 (s), 30.84 (s), 30.77
(s), 30.61 (s), 30.44 (s), 30.35 (s), 30.26 (s), 30.16 (s), 30.13 (s),
28.14 (s), 28.13 (s), 23.72 (s), 14.55 (s). δF(CDCl3): -208.60
(0.5F, m), -210.99 (0.5F, m). δP(CDCl3): 16.21 (0.5P, d, J )
72.7 Hz), 15.95 (0.5P, d, J ) 73.8 Hz). MS (CI) m/z 435.3 (M+
- OH, 60.85), 283.3 (M+ - C4H9 - CFH3PO3, 100.00). HRMS
for C22H41FO5P (M+ - OH): found 435.2678, calcd 435.2676.
Dieth yl [1-flu or o-3(R)-4-d ih yd r oxybu tyl]p h osp h on a te
(24b) was obtained as above as a colorless liquid. δH(CDCl3):
4.97-4.72 (m, 1H), 4.21-4.12 (m, 4H), 3.14-3.10 (m, 1H), 2.83
(t, J ) 4.0 Hz, 0.5H), 2.75 (t, J ) 4.0 Hz, 0.5H), 2.54 (m, 1H),
2.29-2.08 (m, 2H), 1.32 (m, 6H). δC(CDCl3): 86.17 (dd, J )
180.2, 172. 6 Hz), 85.92 (dd, J ) 180.9, 172.5 Hz), 63.35 (d, J
) 3.1 Hz), 63.28 (d, J ) 3.1 Hz), 63.00 (d, J ) 4.6 Hz), 62.93
(d, J ) 4.6 Hz), 48.49 (dd, J ) 14.6, 3.8 Hz), 48.26 (dd, J )
17.6, 3.8 Hz), 47.63 (s), 46.41 (s), 37.80 (d, J ) 19.8 Hz), 32.85
(d, J ) 19.9 Hz), 16.40 (d, J ) 12.4 Hz), 16.35 (d, J
)
12.0 Hz). δF(CDCl3): -207.73 (0.5F, m), -211.17 (0.5F, m).
δP(CDCl3): 19.91 (0.5P, d, J ) 75.0 Hz), 19.40 (0.5P, d, J )
76.1 Hz). [R]20 +16.30 (c 4.50, MeOH).
D
Dieth yl [1-d iflu or o-3(S)-3,4-ep oxy-bu tyl]p h osp h on a te
(25b) was recovered in resolved form as a colorless liquid.
δH(CDCl3): 4.97-4.72 (m, 1H), 4.21-4.12 (m, 4H), 3.14-3.10
(m, 1H), 2.83 (t, J ) 4.0 Hz, 0.5H), 2.75 (t, J ) 4.0 Hz, 0.5H),
2.54 (m, 1H), 2.29-2.08 (m, 2H), 1.32 (m, 6H). δC(CDCl3):
85.92 (dd, J ) 180.9, 172.5 Hz), 86.17 (dd, J ) 180.2, 172. 6
Hz), 63.35 (d, J ) 3.1 Hz), 63.28 (d, J ) 3.1 Hz), 63.00 (d, J )
4.6 Hz), 62.93 (d, J ) 4.6 Hz), 48.49 (dd, J ) 14.6, 3.8 Hz),
48.26 (dd, J ) 17.6, 3.8 Hz), 47.63 (s), 46.41 (s), 37.80 (d, J )
19.8 Hz), 32.85 (d, J ) 19.9 Hz), 16.40 (d, J ) 12.4 Hz), 16.35
(d, J ) 12.0 Hz). δF(CDCl3): -207.73 (0.5F, m), -211.17 (0.5F,
[R]20 -2.13 (c 0.14, MeOH). The compound was converted to
D
the sodium salt by ion exchange and stored in solid form at
-80 °C as described for 1a .
[1-F lu or o-3(S)-h yd r oxyl-4-(p a lm it oyloxy)b u t yl]p h os-
p h on a te (3a b) was obtained similarly from precursor 26a b
in 91% yield. δH(CD3OD): 5.27-5.18 (m, 1H), 4.78 (m, 1H),
3.68 (dd, J ) 10.80, 4.00 Hz, 1H), 3.57 (m, 1H), 2.40-2.25 (m,
4H), 1.64 (m, 2H), 1.33-1.22 (m, 24H), 0.89 (t, J ) 7.2 Hz,
3H). δC(CDCl3): 172.33 (s), 172.30 (s), 87.06 (dd, J ) 170.3,
168.7 Hz), 85.29 (dd, J ) 170.3, 168.7 Hz), 69.33 (dd, J ) 14.2,
2.4 Hz), 67.56 (dd, J ) 14.2, 2.4 Hz), 33.04 (d, J ) 7.7 Hz),
31.92 (s), 31.06 (s), 28.77 (s), 28.75 (s), 28.71 (s), 28.58 (s), 28.47
(s), 28.39 (s), 28.15 (s), 24.05 (s), 23.97 (s), 23.92 (s), 21.72 (s),
12.48 (s). δF(CDCl3): -208.73 (0.5F, m), -211.07 (0.5F, m).
δP(CDCl3): 16.21 (0.5P, d, J ) 72.7 Hz), 15.95 (0.5P, d, J )
73.8 Hz). MS (CI) m/z 409.2 (M+ + 1 - OH - CH3, 2.29),
225.2 (M+ - C14H29 - OH, 100.00). HRMS for C20H38FO5P (M+
m). δP(CDCl3): 18.07 (d, J ) 73.8 Hz). [R]20 +12.06 (c 2.33,
D
MeOH).
Dieth yl [1-F lu or o-3(S)-h yd r oxyl-4-(oleoyloxy)bu tyl]-
p h osp h on a te (26a a ). To a solution of diol 24a (107 mg, 0.438
mmol) and oleic acid (118 mg, 0.416 mmol) in dry CH2Cl2 (2
mL) was added a solution of DCC (109 mg, 0.526 mmol) and
DMAP (32 mg, 0.263 mmol) in dry CH2Cl2 (1 mL) at 0 °C. The
solution was stirred for 16 h at 0 °C, filtered, concentrated in
vacuo, and the residue was purified on silica gel (n-hexanes-
ethyl acetate, HE:AE 1:1, Rf 0.29) to afford ester (121 mg, 0.238
mmol, 51%) as a waxy solid. δH(CDCl3): 5.29 (m, 2H), 5.10-
4.89 (m, 1H), 4.22-3.98 (m, 7H), 3.48 (br, 1H), 2.29 (t, J ) 7.6
Hz, 2H), 2.18-2.03 (m, 2H), 1.93 (m, 4H), 1.58 (m, 2H), 1.33-
1.22 (m, 28H), 0.83 (t, J ) 7.2 Hz, 3H). δC(CDCl3): 173.84 (s),
173.81 (s), 129.92 (s), 129.64 (s), 86.49 (dd, J ) 171.0, 172.6
Hz), 84.71 (dd, J ) 171.1, 172.6 Hz), 68.06 (s), 67.48 (s), 66.01
(dd, J ) 10.0, 3.8 Hz), 65.07 (dd, J ) 13.1, 3.0 Hz), 63.55 (d, J
) 6.9 Hz), 63.30 (d, J ) 6.9 Hz), 63.06 (d, J ) 6.9 Hz), 62.98
(d, J ) 8.4 Hz), 34.36 (d, J ) 19.9 Hz), 33.81 (d, J ) 18.4 Hz),
31.82 (s), 29.67 (s), 29.61 (s), 29.43 (s), 29.23 (s), 29.09 (s), 27.13
(s), 27.08 (s), 24.86 (s), 22.59 (s), 16.35 (m), 14.02 (s). δF(CDCl3):
-208.26 (0.5F, m), -211.75 (0.5F, m). δP(CDCl3): 19.36 (0.5P,
d, J ) 73.8 Hz), 19.10 (0.5P, d, J ) 76.1 Hz). MS (CI) m/z 509.4
- OH - CH3): found 408.2432, calcd 408.2441. [R]20 -1.83
D
(c 0.17, MeOH). The compound was converted to the sodium
salt by ion exchange and stored in solid form at -80 °C as
described for 1a .
Diet h yl [1-flu or o-3(R)-h yd r oxyl-4-(oleoyloxy)b u t yl]-
p h osp h on a te (26ba ) was obtained as a waxy solid in 56%
yield. δH(CDCl3): 5.29 (m, 2H), 5.10-4.90 (m, 1H), 4.22-3.98
(m, 7H), 3.44 (br, 1H), 2.30 (t, J ) 7.6 Hz, 2H), 2.18-2.03 (m,
2H), 1.93 (m, 4H), 1.56 (m, 2H), 1.33-1.22 (m, 28H), 0.83 (t, J
) 7.2 Hz, 3H). δC(CDCl3): 173.84 (s), 173.81 (s), 129.92 (s),
129.64 (s), 86.49 (dd, J ) 171.0, 172.6 Hz), 84.71 (dd, J ) 171.1,
172.6 Hz), 68.06 (s), 67.48 (s), 66.01 (dd, J ) 10.0, 3.8 Hz),
65.07 (dd, J ) 13.1, 3.0 Hz), 63.55 (d, J ) 7.0 Hz), 63.30 (d, J
(M+ + 1, 29.75), 463.3 (M+ - OC2H5, 100.00). HRMS for C26H51
-
J . Org. Chem, Vol. 68, No. 13, 2003 5329

Xu et al.
) 7.0 Hz), 63.06 (d, J ) 7.0 Hz), 62.98 (d, J ) 8.4 Hz), 34.36
(d, J ) 19.9 Hz), 33.81 (d, J ) 18.4 Hz), 31.82 (s), 29.67 (s),
29.61 (s), 29.43 (s), 29.23 (s), 29.09 (s), 27.13 (s), 27.08 (s), 24.86
(s), 22.59 (s), 16.35 (m), 14.02 (s). δF(CDCl3): -208.29 (0.5F,
m), -211.75 (0.5F, m). δP(CDCl3): 19.36 (0.5P, d, J ) 73.8 Hz),
min, and then cooled to -20 °C. Aldehyde 28 (3.3 g, 22.92
mmol) in 20 mL of THF was transferred into the solution at
this temperature. The reaction mixture was allowed to warm
to room temperature slowly and stirred overnight and then
quenched by slow addition of acetic acid (24.1 mmol, 1.39 mL)
in 10 mL of ether. The organics were filtered through Celite,
which were then washed with ethyl acetate. The solvents were
removed in vacuo to give a colorless oil that was purified by
19.10 (0.5P, d, J ) 76.1 Hz). [R]20 +2.47 (c 1.86, MeOH).
D
Dieth yl[1-flu or o-3(R)-h yd r oxyl-4-(p a lm itoyloxy)bu tyl]-
p h osp h on a te (26bb) was obtained as a white solid in 53%
yield. δH(CDCl3): 5.11-4.90 (m, 1H), 4.20-3.99 (m, 7H), 3.42
(br, 1H), 2.29 (t, J ) 7.6 Hz, 2H), 2.19-1.90 (m, 2H), 1.58 (t,
J ) 6.8 Hz, 2H), 1.33 (t, J ) 6.8 Hz, 6H), 1.60 (m, 24H), 0.83
(t, J ) 7.2 Hz, 3H). δC(CDCl3): 173.88 (s), 173.85 (s), 86.00
(dd, J ) 178.7, 171.1 Hz), 85.23 (dd, J ) 178.7, 171.1 Hz), 68.06
(s), 67.50 (s), 66.05 (dd, J ) 10.1, 4.6 Hz), 65.08 (dd, J ) 10.1,
4.6 Hz), 63.44 (dd, J ) 25.3, 7.6 Hz), 63.04 (dd, J ) 6.8, 6.8
Hz), 34.37 (d, J ) 19.9 Hz), 31.85 (s), 29.61 (s), 29.57 (s), 29.53
(s), 29.38 (s), 29.28 (s), 29.19 (s), 29.07 (s), 22.61 (s), 16.38 (d,
J ) 5.3 Hz), 16.34 (d, J ) 4.6 Hz), 14.03 (s). δF(CDCl3): -208.28
(0.5F, m), -211.75 (0.5F, m). δP(CDCl3): 19.37 (0.5P, d, J )
1
flash chromatography to afford the phosphonate 29. H, 13C,
31P NMR are identical with those reported.50
1-Dieth ylp h osp h on yl-1-flu or in e-3,4-O-isop r op ylid en e-
1(R,S)-3(S),4-bu ta n etr iol (30) was prepared by DAST fluo-
rination following the procedure described for compound 16.
δH(CDCl3): 4.70-5.01 (m, 1H), 4.04-4.35 (m, 6H), 3.54-3.66
(m, 1H), 1.90-2.28 (m, 2H), 1.30-1.38 (m, 12H). δP (CDCl3),
18.65 (d, J ) 73.84 Hz, integration, 91.42), 18.36 (d, J ) 76.10
Hz, integration, 8.58). δF(CDCl3): -207.52 (0.085F, m), -212.52
(0.915F, m).25
P la telet Activa tion Assa ys. Selected compounds were
tested at a concentration of 1 µM for their ability to activate
gel-filtered platelets (∼300 000 platelets/µL in 129 mM NaCl,
9.9 mM NaHCO3, 2.8 mM KCl, 0.8 mM KH2PO4, 5.6 mM
dextrose, 10 mM HEPES, 2 mM CaCl2, 1 mM MgCl2) in the
presence of fibrinogen (100 µg/mL), using a dual channel
aggregometer (Chronolog; Haverton, PA) as previously de-
scribed.53
73.8 Hz), 19.10 (0.5P, d, J ) 76.1 Hz). [R]20 +3.01 (c 0.84,
D
MeOH).
[1-F lu or o-3(R)-h yd r oxyl-4-(oleoyloxy)bu tyl]p h osp h o-
n a te (3ba ) was obtained in 94% yield from precursor 26ba .
δH(CD3OD): 5.34 (m, 2H), 5.33-5.17 (m, 1H), 4.79 (m, 1H),
3.68 (dd, J ) 11.60, 4.40 Hz, 1H), 3.59 (m, 1H), 2.35 (m, 4H),
2.02 (m, 4H), 1.61 (m, 2H), 1.33-1.22 (m, 20H), 0.89 (t, J )
7.2 Hz, 3H). δC(CDCl3): 174.38 (s), 174.22 (s), 130.84 (s), 130.74
(s), 88.16 (dd, J ) 170.25, 168.74 Hz), 86.39 (dd, J ) 170.25,
168.74 Hz), 71.30 (dd, J ) 14.58, 2.31 Hz), 69.52 (dd, J ) 14.58,
2.31 Hz), 35.12 (d, J ) 19.32 Hz), 34.93 (d, J ) 18.89 Hz),
33.04 (s), 30.84 (s), 30.77 (s), 30.61 (s), 30.44 (s), 30.35 (s), 30.26
(s), 30.16 (s), 30.13 (s), 28.14 (s), 28.13 (s), 23.72 (s), 14.55 (s).
δF(CDCl3): -208.68 (0.5F, m), -210.99 (0.5F, m). δP(CDCl3):
16.01 (0.5P, d, J ) 72.86 Hz), 15.93 (0.5P, d, J ) 74.00 Hz).
P P ARγ Activa tion Assa ys.5 RAW 264.7 monocytic cells
were transfected with rat acyl-CoA oxidase PPRE-luciferase
and SV40 â-galactosidase reporter plasmids, and treated with
5 µM sn-1-O-oleoyl LPA (Avanti Polar Lipids, Alabaster, AL)
or with 5 µM of each of the four sn-1 or sn-2-monofluoro
analogues of LPA for 16 h. Then, relative amounts of luciferase
and â-galactosidase were determined. Data are shown as
relative light units (luciferase) normalized for transfection
efficiency (â-Gal).
[R]20 +2.01 (c 0.22, MeOH). The compound was converted to
D
the sodium salt by ion exchange and stored in solid form at
-80 °C as described for 1a .
Ack n ow led gm en t. We thank the Utah Centers of
Excellence Program and the Human Frontier Science
Program (RG0073-2000-B) for financial support of this
work. We thank Drs. A. J . Morris and S. Smyth
(University of North Carolina) for permission to cite the
platelet activation results, and Drs. A. Ponstler and T.
M. McIntyre (The University of Utah) for permission
to cite the PPARγ ligand activation data. We also thank
Drs. J . Aoki and K. Hama (University of Toyko) for
permission to cite unpublished results on cell-based LPA
receptor activation results, which were presented in
poster form on April 11, 2003.
[1-F lu or o-3(R)-h yd r oxyl-4-(p a lm it oyloxy)bu t yl]p h os-
p h on a te (3bb) was obtained in 88% yield from precursor
26bb. δH(CD3OD): 5.27-5.18 (m, 1H), 4.78 (m, 1H), 3.68 (dd,
J ) 10.80, 4.00 Hz, 1H), 3.57 (m, 1H), 2.40-2.25 (m, 4H), 1.64
(m, 2H), 1.33-1.22 (m, 24H), 0.89 (t, J ) 7.2 Hz, 3H).
δC(CDCl3): 172.33 (s), 172.30 (s), 87.06 (dd, J ) 170.25, 168.74
Hz), 85.29 (dd, J ) 170.25, 168.74 Hz), 69.33 (dd, J ) 14.21,
2.35 Hz), 67.56 (dd, J ) 14.21, 2.35 Hz), 33.04 (d, J ) 7.68
Hz), 31.92 (s), 31.06 (s), 28.77 (s), 28.75 (s), 28.71 (s), 28.58
(s), 28.47 (s), 28.39 (s), 28.15 (s), 24.05 (s), 23.97 (s), 23.92 (s),
21.72 (s), 12.48 (s). δF(CDCl3): -208.73 (0.5F, m), -211.07
(0.5F, m). δP(CDCl3): 16.19 (0.5P, d, J ) 72.70 Hz), 15.84 (0.5P,
d, J ) 73.84 Hz). [R]20 +2.56 (c 0.13, MeOH). The compound
D
was converted to the sodium salt by ion exchange and stored
in solid form at -80 °C as described for 1a .
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectra of
all the intermediates and the monofluorinated LPA analogues
(1, 2, 3) described herein. This material is available free of
charge via the Internet at http://pubs.acs.org.
1-Dieth ylph osph on yl-3,4-O-isopr opyliden e-1(R,S)-3(S),4-
bu ta n etr iol (29). To a solution of diethyl phosphite (3.80 g,
24.1 mmol) in 8 mL of THF at -78 °C was added lithium bis-
(trimethylsilyl)amide (24.1 mL, 1.0 M) in THF. The solution
was allowed to warm to room temperature and stirred for 45
J O020729L
5330 J . Org. Chem., Vol. 68, No. 13, 2003