Phosphonothioate and fluoromethylene phosphonate analogues of cyclic phosphatidic acid: Novel antagonists of lysophosphatidic acid receptors

Article abstract of DOI:10.1021/jm060351+

Isoform-selective antagonists of the lysophosphatidic acid (LPA) G-protein coupled receptors (GPCRs) have important potential uses in cell biology and clinical applications. Novel phosphonothioate and fluoromethylene phosphonate analogues of carbacyclic p

Full text of DOI:10.1021/jm060351+

J. Med. Chem. 2006, 49, 5309-5315
5309
Phosphonothioate and Fluoromethylene Phosphonate Analogues of Cyclic Phosphatidic Acid:
Novel Antagonists of Lysophosphatidic Acid Receptors§
Yong Xu,† Guowei Jiang,† 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
ReceiVed March 26, 2006
Isoform-selective antagonists of the lysophosphatidic acid (LPA) G-protein coupled receptors (GPCRs) have
important potential uses in cell biology and clinical applications. Novel phosphonothioate and fluoromethylene
phosphonate analogues of carbacyclic phosphatidic acid (ccPA) were prepared by chemical synthesis. The
pKa values of these amphilic phosphonolipids and the parent cyclic phosphonate were measured titrimetrically
using the Yasuda-Shedlovsky extrapolation. The pharmacological properties of these and other ccPA
analogues were characterized for LPA receptor (LPAR) subtype-specific agonist and antagonist activity
using Ca2+-mobilization assays in RH7777 cells expressing the individual EDG-family GPCRs. In particular,
the phosphonothioate ccPA analogue inhibited Ca2+ release through LPA1/LPA3 activation and was an LPA1/
LPA3 antagonist. The monofluoromethylene phosphonate ccPA analogue was also a potent LPA1/LPA3
antagonist. In contrast, the difluoromethylene phosphonate ccPA analogue was a weak LPAR agonist, while
ccPA itself had neither agonist nor antagonist activity.
Introduction
The unique Physarum phospholipid PHYLPA (Figure 1),
isolated from myxoamoebae of Physarum polycephalum, is an
inhibitor of eukaryotic DNA polymerase.1 PHYLPA has a
cyclopropane-containing fatty acyl chain and also possesses a
cyclic phosphate at sn-2 and sn-3 positions of the glycerol
carbons.1,2 PHLYPA inhibits the proliferation of human fibro-
blasts cultured in a chemically defined medium.3 Although this
compound is referred to as a cyclic phosphatidic acid (cPA)
analogue, the absence of the second acyl group makes it more
accurately a cyclic form of lysophosphatidic acid (LPA). The
biological activities of synthetic analogues of PHYLPA4 and
the structure-activity relationships (SARs) indicated that the
cyclic phosphate was required for antiproliferative activity.5 The
cellular effects elicited by cPA include antimitogenic regulation
of the cell cycle,3,5,6 regulation of actin stress fiber formation
and rearrangement,6 inhibition of cancer cell invasion and
metastasis,7 inhibition of platelet aggregation,8 regulation of
differentiation and viability of neuronal cells, activation of Cl-
currents,9 and mobilization of intracellular calcium.3 cPA is
generated in blood and is bound to serum albumin.10 Recently,
a metabolically stabilized carbacyclic analogue of cPA, known
as ccPA, was described as a novel inhibitor of metastatic cancer
(Figure 1).11,12
In exploring a wide range of metabolically stabilized LPA
analogues,13 we have identified the phosphorothioate14,15 and
mono- and difluoromethylene phosphonate moieties16,17as im-
portant bioactive mimics of the phosphate. We thus envisaged
the synthesis of the corresponding cyclic phosphonothioate and
mono(di)fluoromethylene phosphonate analogues of ccPA (Fig-
ure 1). On the basis of studies of second pKa values for acyclic
phosphomonoesters,18 these analogues were expected to have
pKa values more closely matched the parent cyclic phosphate
Figure 1. Cyclic phosphatidic acids and their carbacyclic analogues.
than the cyclic methylenephosphonate ccPA. Thus, in addition
to retaining the metabolic stability of the carbacylic structure,
we anticipated that these ccPA analogues would have improved
receptor binding properties. Since LPA receptors (LPARs) are
responsible for cell proliferation and differentation, cell migra-
tion, and cell invasion, we hypothesized that these ccPA
analogues could be potential LPAR antagonists.19,20 Herein, we
describe the synthesis of novel ccPA and their activities on
human LPAR subtypes.20-22
Phosphonothioate, or phosphothioic acids, and their deriva-
tives have generated considerable interest as inhibitors of
enzymes involved in metabolism of the natural phosphates.
Phosphonothioates thus have potential applications for the
selective manipulation of fundamental cellular responses that
could validate new therapeutic approaches for human diseases.23
One common approach for stabilization of phosphate monoesters
is the use of a mono- or difluoromethylene phosphonate
analogues in place of the higher pKa methylene or methyl
phosphonates. Though less well studied, recent experimental
and theoretical reports have suggested that the R-fluorometh-
ylene phosphonate groups should be able to better mimic the
phosphate than either the methylene or difluoromethylene
derivatives.18,24 Several monofluoromethylene phosphonates
have been studied as potential enzyme inhibitors and as probes
for the elucidation of biochemical processes.25 Indeed, we have
§ Dedicated to Professor Iwao Ojima on the occasion of his 60th birthday.
* Corresponding author. Fax: 01-801-585-9053. email: gprestwich@
pharm.utah.edu.
† The University of Utah.
‡ University of Tennessee Health Science Center.
10.1021/jm060351+ CCC: $33.50 © 2006 American Chemical Society
Published on Web 07/27/2006

5310 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17
Scheme 1. Synthesis of the Cyclic Phosphonothioatesa
Xu et al.
Scheme 2. Synthesis of the Cyclic Mono- and
Difluorophosphonatesa
a Reagents and conditions: (a) TMSBr, CH2Cl2; CH3OH/H2O, 75-93%;
(b) DCC, DMF; (c) RCOCl, pyridine, DMF, 51%.
a Reagents and conditions: (a) LiCH2P(O)(OCH3)2, THF, BF3.Et2O,
100%; (b) PPTS, toluene, 44%; (c) 10% Pd/C, 1 atm H2, 78%; (d) TBSCl,
imidazole, DMF, 97%; (e) Lawesson’s reagent, toluene, 67%; (f) TBAF‚3H2O,
HOAc, THF, 69%; (g) RCOOH, DCC/DMAP, CH2Cl2, 73-86%; (h)
t-BuNH2, 71%.
with TBAF that had been neutralized with acetic acid, affording
the primary alcohol 7. Esterification (DCC, DMAP, and either
oleic acid or palmitic acid, rt) provided the racemic esters 8a
and 8b, respectively.
Deprotection of the methyl ester of the cyclic phosphono-
thioate required a careful choice of reagent. While the commonly
used electrophiles TMSBr or TMSI fail to deprotect phospho-
nothioates, tert-butylamine,33 trimethylamine,34 and lithium in
liquid ammonia34 have been used for similar reactions. We
selected tert-butylamine, because it was reported to deprotect
methyl esters of either phosphonothioates or phosphorothioates
under mild conditions. In the event, each of the charged
phosphonothioates 9 was obtained by refluxing the protected
esters 8 in tert-butylamine for 3 days.
R-Monofluoroalkylphosphonates can be prepared in modest
yields by electrophilic fluorination of R-carbanions of alkyl
phosphonate esters with NFSI or Selectfluor.35 Consequently,
we reasoned that electrophilic fluorination might be a viable
method for the preparation of R-monofluoroalkylphosphonic
acids. However, attempts at electrophilic fluorination of the
cyclic phosphonate anion with NFSI and Selectfluor at low
temperatures were unsuccessful using a variety of bases (NaH,
LDA, KDA and n-BuLi) and solvents (DMF, THF). An alternate
approach was thus required.
recently described the enantioselective synthesis17,26,27 and
selected biological activities of a variety of fluorinated analogues
of LPA.13,16 The biological results demonstrated that R-monof-
luorinated phosphonate mimics of LPA were significant phos-
phatase-resistant and isoform-selective agonists for LPARs.
Chemical Synthesis
Current methods for the preparation of phosphonothioates
were developed for specific compounds and employed condi-
tions that would preclude application to the synthesis of labile
LPA analogues. For example, methyl and ethyl esters have often
been used as protecting groups during syntheses of phosphatidic
and phosphonatidic acids. However, deprotection with trimeth-
ylsilyl iodide or trimethylsilyl bromide gave low yields of
phosphonothioate or phosphorothioate derivatives.28 Deprotec-
tion of the dibenzyl esters of phosphonothioic acids with sodium
in liquid ammonia29 and deprotection of the cyanoethyl esters
under mild basic conditions14 are presently the most frequently
used methods for the synthesis of phosphonothioates and
phosphothioates.
We previously described the addition of diethyl iodomonof-
luoro- and iododifluoromethylene phosphonates to allyl alcohol
catalyzed by tetrakis(triphenylphosphine)palladium in hexane
to give the corresponding iodohydrins in 72% yield.17,27 The
epoxides were formed by treatment with K2CO3-MeOH for 5
min at room temperature and then subjected to a hydrolytic
kinetic resolution (HKR) reaction with 0.45 equiv of H2O in a
minimum volume of THF in the presence of 1.0 mol % of (R,R)-
Salen-Co-OAc.17,27 The diols 10a and 10b were obtained in 99%
ee. When neither p-TsOH or PPTS could cyclize the phospho-
nate, we turned to dicyclohexylcarbondiimide (DCC), which
had been reported to promote efficient intramolecular, high-
dilution cyclization of phosphates or phosphonates, which
generated the corresponding cyclic analogues.36-38 When the
formation of either five- and six-membered cyclic phosphates
is possible, the five-membered ring was favored for both the
phosphate or phosphonate products. For example, R-glycero-
phosphate formed a five-membered ring preferentially upon
treatment with DCC.37 The diols 11a and 11b were prepared
readily by treatment with TMSBr (Scheme 2).39,40 Thus, reaction
of diol 11a with DCC was followed immediately by esterifi-
cation of the remaining hydroxy group. NMR data established
the cyclization product 13 indeed possessed the desired five-
membered ring. Compound 13a had a double doublet resonance
at 31-32 ppm in the 31P NMR, while the typical acyclic
We sought a convenient and versatile synthetic route to
phosphonothioate ccPA. Regiospecific ring opening of the
commercially available benzyl glycidol ether with a carbon
nucleophile, derived from dimethyl methylphosphonate in the
presence of BF3‚OEt2, furnished the corresponding racemic
γ-hydroxy phosphonate 2 (Scheme 1).30 An intramolecular
transesterification reaction of 4-(benzyloxy)-3-hydroxybutane-
phosphonate to the corresponding cyclic compound 3 was
achieved with pyridinium p-toluenesulfonate (PPTS) in toluene.
With the cyclic intermediate in hand, it was important to conduct
the hydrogenolysis of the benzyl protecting group prior to
thionation of phosphonate.
Thus, hydrogenolysis (Pd-C, 1 atm H2) proceeded readily
at room temperature (rt). The primary alcohol 4 was protected
as a tert-butyldimethylsilyl (TBS) ether 5, which can survive
thionation and yet still be removed under mild conditions.
Introduction of the PdS group is the key transformation for
the synthesis of phosphonothioates and phosphorothioates. We
chose Lawesson’s reagent for this transformation, as improved
yields of thiophosphoryl products and milder reaction conditions
31
were reported in comparison to P2S5.32 Thus, thionation of
cyclic phosphonate 5 with Lawesson’s reagent was accom-
plished by refluxing in toluene for 8 h to give the desired cyclic
phosphonothioate 6 in good yield. The TBS ether 6 was removed

Antagonists of Lysophosphatidic Acid Receptors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17 5311
Table 1. Aqueous pKa Values of CcPA and Its Analogues 9a, 9b, 13a,
and 13b Measured in Water-Methanol Mixtures and Extrapolated to
Zero Methanol Contenta
1-oleoyl LPA. Importantly, the phosphonothioate ccPA ana-
logues did not show any agonist-related activitation of the three
LPA GPCRs tested.
compound
pKa value
Our observation that ccPA was not an antagonist on LPA
GPCRs suggested that the inhibition of metastasis was not due
to a direct effect on LPARs,12 and that ccPA must have
additional targets mediating anti-metastatic effect. Intriguingly,
the simple substitution of a PdO bond with a PdS bond in the
phosphonothioate resulted in a potent LPA1/LPA3 antagonist.
This effect cannot be due solely to matching the pKa values,
which were very similar for ccPA and the phosphonothioate
analogues 9. In addition, the monofluoromethylene phosphonate
ccPA analogue 13a, with a lower pKa value of 5.60, was
observed to be a potent LPA1/LPA3 antagonist. Surprisingly,
this antagonist activity was abrogated upon introduction of a
second fluorine atom in analogue 13b, which lowered the pKa
value to 4.62. The difluoromethylenephosphonate 13b was now
a weak LPAR agonist. Clearly, the number of fluorine atoms
at the R-position of the cyclic phosphonate controls both the
pKa and the nature of the ligand-receptor interaction.
The ccPA structure appears to offer a promising scaffold for
the design of new LPAR antagonists with selective interactions
with receptors. Selective and potent LPAR antagonists are highly
desirable for dissecting the importance of LPAR function in
cell biology,13,19,20 and recent computational studies now offer
avenues to incorporate selective design strategies.19,45 In addi-
tion, LPAR antagonists may have therapeutic potential to control
abnormal cell growth, differentiation, and inflammation in
cancer and vascular pathophysiology.20-22
ccPA
9a
9b
13a
13b
6.58 ( 0.07
6.76 ( 0.05
6.48 ( 0.01
5.60 ( 0.01
4.62 ( 0.09
a See text for experimental details.
monofluoromethylene phosphonate would have resonances at
ca. 20 ppm. 1H NMR spectra showed a 3-H resonance at 4.49-
4.38 ppm; the chemical shift of 3-H in the six-membered ring
would be expected to occur above 5.2 ppm.26
pKa Determination of ccPA and Its Analogues in Metha-
nol-Water Mixture. Acid dissociation constants (pKa values)
are important in pharmaceutical drug design and discovery,
where knowledge of the ionization state of a particular functional
group is often vital in order to understand the pharmacokinetic
and pharmacodynamic properties of new entities. Ionizable
groups in aqueous solution can be determined readily by pH-
metric titration, but poorly water-soluble compounds are gener-
ally unsuitable for pH-metric titration. However, if the com-
pound is sufficiently soluble in a water-miscible organic solvent,
it is possible to determine the apparent pKa (psKa) pH-metrically
in cosolvent mixtures. Aqueous pKa values can be then
determined by extrapolation of the psKa values to zero organic
solvent content.41,42
The apparent acid dissociation constants (psKa) of ccPA and
analogues 9a, 9b, 13a, and 13b were determined pH-metrically
in methanol-water mixtures. A glass electrode calibration
procedure based on a four-parameter equation (pH ) R + SpcH
+ jH[H+] + jOH[OH-]) was used to obtain pH readings based
on the concentration scale (pcH). The Yasuda-Shedlovsky
extrapolation (psKa + log [H2O] ) A/ꢀ + B) was used to derive
acid dissociation constants in aqueous solution (pKa).41,42 The
measured pKa values are shown in Table 1. Interestingly, ccPA
and the two phosphonothioates 9 had very similar pKa values
in the range of 6.46-6.78. The monofluoromethylenephospho-
nate 13a was one pKa unit more acidic at 5.60, while difluo-
romethylenephosphonate analogue 13b showed the lowest pKa
value (4.62) as expected from the strong electron-withdrawing
ability of the difluoromethylene group.
Receptor Activation Studies. The ligand properties of the
compounds were evaluated on the activation of LPARs designed
(by convention) LPA1, LPA2, and LPA3 by Ca2+ mobilization
in RH7777 cells expressing each individual human LPAR
subtype. Table 2 illustrates calcium responses elicited through
the activation of human LPA1, LPA2, and LPA3 receptors. In
these experiments, RH7777 cells, which are intrinsically
unresponsive to LPA, were transfected with human LPA1, LPA2,
and LPA3, respectively.43,44 While ccPA itself had no detectable
LPA receptor agonist or antagonist effects, the difluorometh-
ylene phosphonate ccPA 13b was a weak LPA receptor agonist
at 10 µM, compared to an EC50 value for natural sn-1-oleoyl
LPA of 50-100 nM for the different LPA receptor isoforms.
Thus, 13b was >100-fold less potent than natural LPA.
In contrast, the monofluoromethylene phosphonate ccPA 13a
was an LPA1/LPA3 antagonist. For LPA1, 13a was a partial
antagonist, with a maximal inhibition of 46% of the response
induced by 200 nM sn-1-oleoyl LPA. Interestingly, both the
phosphonothioate ccPA analogues 9a and 9b were also LPA1/
LPA3 selective antagonists. The oleyl analogue 9a was a partial
LPA1 antagonist, blocking 64% of the response to 200 nM sn-
Recent evidence shows that LPA is produced extracellularly
from lysophosphatidylcholine by the lysophospholipase D
activity of autotaxin (ATX/lysoPLD).46 This enzyme is a
ubiquitous exo-phosphodiesterase that was originally identified
as an autocrine motility factor for melanoma cells and has been
implicated in tumor progression. The local production of LPA
by ATX/lysoPLD could support an invasive microenvironment
for tumor cells, exacerbating metastasis.22 Thus, LPAR antago-
nists and ATX inhibitors may have therapeutic potential in
cancer therapy by blocking the growth-supporting and antiapo-
ptotic effects of LPA and reducing its titer. The mechanisms of
enhanced tumor cell invasion by LPA include two important
molecular mechanisms. First, LPAR-mediated activation of the
Rho and Rac GTPase pathways are essential for the regulation
of the actin cytoskeleton and cell motility.47 Second, LPA has
been shown to regulate the activity of matrix metalloproteinases,
which are also intricately involved in metastasis as well as in
the LPA-induced transphosphorylation of the epidermal growth
factor (EGF) receptor.48
Currently available LPAR antagonists represent a promising
start to the development of useful chemical tools, although none
can be considered definitive in determining receptor selectivity
or biological functions, especially for studies in vivo. Several
LPAR antagonists have been reported. A list of compounds with
reported LPAR selectivity includes the ethanolamide phosphate
(VPC12249, LPA1/LPA3 antagonist),49,50 3-(4-[4-([1-(2-chlo-
rophenyl)-ethoxy]carbonylamino)-3-methyl-5-isoxazolyl]benzyl-
sulfanyl)propanoic acid (Ki16425, LPA1/LPA3 antagonist),51
decyl fatty alcohol phosphates (FAP-10, LPA1/LPA3 antagonist
and LPA2 agonist),44 diacylglyceryl pyrophosphate (DGPP, an
LPA1/LPA3 antagonist).43 Appropriately validated compounds
are essential to advance in vivo studies, particularly in view of
potential off-target effects. The development of more selective,
more stable, more potent, and more drug-like antagonists is

5312 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17
Xu et al.
Table 2. Effects of ccPA Analogues 9a, 9b, 13a, and 13b on RH7777 Cells Expressing LPA1-LPA3
LPA1
IC50 (nM)
NE c
LPA2
Emax (%)
NA
NA
NA
NA
LPA3
IC50 (nM)
NE
1270 ( 160 636 ( 80
2340 ( 150 1150 ( 73.3
7720 ( 1740 3170 ( 713
compd EC50 (nM) Emaxb (%)
Ki (nM)
NA d
EC50 (nM)
IC50 (nM) Ki (nM) EC50 (nM)
Emax (%)
NA
NA
NA
NA
Ki
ccPA
9a
9b
13a
13b
NE
NE
NE
NE
NA
NA
NA
NA
NE
NE
NE
NE
NE
NE
NE
NE
NE
NA
NA
NA
NA
NA
NE
NE
NE
NE
NA
941 ( 197 e 403 ( 84.5
799 ( 197 314 ( 77.5
106 ( 58 f
13.0 ( 0.66 NE
60.7 ( 33.5
NA
>1940
>9460
82.0 ( 2.27
at 30 µM
>7030
65.4 ( 5.12 NE
NA
at 30 µM
at 30 µM
b
a Data represent the average of four independent measurements (mean ( SD). Emax) maximal efficacy of the drug/maximal efficacy of LPA 18:1 at
saturation. c NE ) no effect at the highest concentration (30 µM) tested. d NA ) not applicable. e Partial antagonist with 64% inhibition of the 200 nM at
30 µM LPA response. f Partial antagonist with 46% inhibition of the 200 nM LPA response at 30 µM.
(m, 1H). 13C NMR (CDCl3): δ 137.6 (s), 128.4 (s), 127.7 (s), 127.7
(s), 127.5 (s), 77.3 (d, J ) 10.0 Hz), 73.5 (s), 72.1 (d, J ) 6.1 Hz),
52.4 (d, J ) 6.9 Hz), 25.8 (s), 18.3 (d, J ) 121.2 Hz). 31P NMR
(CDCl3): δ 51.04 (s). MS (CI) m/z 257.1 (M++1, 100.00). HRMS,
M++1, Found: 257.0980. Calcd for C12H18O4P, 257.1017.
eagerly awaited. The findings presented herein provide a useful
platform for further optimization of ccPA analogues as LPAR
antagonists.
In conclusion, we have demonstrated efficient enantioselective
syntheses of phosphonothioate and fluoromethylene phosphonate
ccPA analogues and their biological activities as LPA1/LPA3
antagonists. These versatile synthetic routes are currently being
employed for the synthesis of other phosphonothioate and
fluoromethylene phosphonate analogues and will be reported
in due course.
Methyl 3,4-Dihydroxybutane 1,3-Cyclic Phosphonate (4). A
solution of 3 (2.1 g, 8.20 mmol) in absolute methanol (100 mL)
containing 10% Pd-C catalyst (0.83 g) was stirred at ambient
temperature under hydrogen (1 atm) until gas uptake ceased (18
h). Filtration and evaporation under reduced pressure gave com-
pound 4, which was purified flash chromatography (acetone-
hexane 2:1, Rf ) 0.18) to give 1.06 g of homogeneous product
(6.40 mmol, 78% yield).1H NMR (CDCl3): δ 4.27 (m, 1H), 3.68-
3.76 (m, 2H), 3.72 (d, J ) 12.0 Hz, 3H), 3.60 (m, 1H), 2.10-2.22
(m, 2H), 2.00 (m, 1H), 1.80 (m, 1H). 13C NMR (CDCl3): δ 79.3
(d, J ) 10.0 Hz), 64.5 (d, J ) 6.1 Hz), 52.5 (d, J ) 6.9 Hz), 24.9
(s), 18.5 (d, J ) 120.7 Hz). 31P NMR (CDCl3): δ 52.11 (s). MS
(CI) m/z 167.0 (M++1, 100.00). HRMS, M++1, Found: 167.0474.
Calcd for C5H12O4P, 167.0475.
Experimental Section
General Procedures. Chemicals were obtained from Aldrich
and Arcos Chemical Corporation and were used without prior
purification. Solvents used were of reagent grade and were distilled
before use. THF was distilled from sodium wire, and methylene
chloride was distilled from CaH2. Reactions were performed under
an inert atmosphere (N2 or Ar) unless otherwise indicated. Flash
chromatography (FC) employed Whatman 230-400 mesh ASTM
silica gel. 1H and 13C spectra were recorded at 400 MHz (1H), 101
MHz (13C), 162 MHz (31P) and 376 MHz (19F) at 25 °C. Chemical
shifts are reported in ppm with TMS as internal standard for 13C
Methyl 3-Hydroxyl-4-tert-Butyldimethylsilyloxybutane 1,3-
Cyclic Phosphonate (5). Alcohol 4 (0.420 g, 2.53 mmol) was
dissolved in anhydrous DMF (10 mL) and stirred with imidazole
(0.206 g, 3.04 mmol, 1.2 equiv) and tert-butyldimethylsilyl chloride
(TBSCl) (0.420 g, 2.78 mmol, 1.1 equiv) for 24 h at room
temperature. The solution was diluted with water (5 mL) and ethyl
acetate (20 mL), and the aqueous layer was separated and extracted
with EtOAc (3 × 20 mL). The combined organic layers were dried
with Na2SO4 and concentrated in vacuo, and the residue was
purified by FC (hexanes-ethyl acetate 2:1, Rf ) 0.40) to afford
0.392 g of the TBDMS ether 5 as a colorless liquid (1.40 mmol,
1
and H (δ ) 0.00); for 31P, 85% H3PO4 was assigned δ ) 0.00;
for 19F, CFCl3 (δ)0.00).
Dimethyl 4-(Benzyloxy)-3-hydroxybutanephosphonate (2). A
2.5 M solution of n-BuLi (60 mL, 150 mmol) in hexane was added
dropwise to a stirred solution of dimethyl methylphosphonate (18.6
g, 16.25 mL, 150 mmol) in dry THF (150 mL) at -78 °C under a
nitrogen atmosphere. After 15 min of stirring, a solution of the
benzyl glycidol ether (8.21 g, 7.65 mL, 50 mmol) in THF (25 mL)
was added dropwise, followed by BF3‚OEt2 (25.35 mL, 200 mmol),
which was slowly introduced, while maintaining the temperature
below -70 °C. After stirring for 2 h, the reaction was quenched
with saturated NH4Cl (150 mL) and was allowed to warm to room
temperature. The residue obtained after concentration in vacuo was
extracted with four portions of EtOAc (200 mL × 4). The organic
extracts were washed with brine, dried with Na2SO4, and concen-
trated, and the residue was purified by FC (acetone/hexane: 1:1,
Rf ) 0.30) to yield 14.4 g of the pure hydroxy phosphonate ester
(50 mmol, 100%). 1H NMR (CDCl3): δ 7.30-7.22 (m, 5H), 4.48
(s, 2H), 4.34 (m, 1H), 3.76 (d, J ) 10.8 Hz, 6H), 3.39 (m, 2H),
2.23 (m, 1H), 2.09 (m, 1H), 1.96 (m, 1H), 1.85 (m, 1H). 31P NMR
(CDCl3): δ 36.60 (s). MS (CI) m/z 289.1 (M++1, 100.00). HRMS,
M++1, Found: 289.1211. Calcd for C13H22O5P, 289.1217.
Methyl 3-Hydroxyl-4-benzyloxybutane 1,3-Cyclic Phospho-
nate (3). To dimethyl 4-(benzyloxy)-3-hydroxybutanephosphonate
(20.2 g, 70.18 mmol) dissolved in anhydrous toluene (450 mL)
was added PPTS (pyridinium p-toluene sulfonate, 34.0 g, 140
mmol). The mixture was heated to 80 °C for 20 h. After cooling to
room temperature, H2O (200 mL) was added, and the mixture was
extracted with ethyl acetate. The organic phase was dried with Na2-
SO4 and concentrated, and the residue was purified by FC (acetone/
hexane: 1:1, Rf ) 0.48) to yield the pure hydroxy phosphonate
1
55%). H NMR (CD3Cl): δ 4.22 (m, 1H), 3.76-3.71 (m, 5H),
2.24-2.06 (m, 2H), 1.97-1.74 (m, 2H), 0.84 (s, 9H), 0.02 (m,
6H). 13C NMR (CD3Cl): δ 78.2 (d, J ) 9.2 Hz), 67.2 (d, J ) 6.1
Hz), 52.1 (d, J ) 6.9 Hz), 25.6 (s), 25.5 (d, J ) 23.0 Hz), 18.7 (d,
J ) 122.7 Hz), 18.1 (s), -5.6 (s), -5.7 (s). 31P NMR (CDCl3): δ
113.43 (s). MS (CI) m/z 281.2 (M++1, 100.00). HRMS, M++1,
Found: 281.1342. Calcd for C11H26O4PSi, 281.1346.
Methyl 3-Hydroxyl-4-tert-butyldimethylsilyloxybutane 1,3-
Cyclic Phosphonothioate (6). A solution of 5 (0.553 g, 1.98 mmol)
and Lawesson’s Reagent (2,4-bis(p-methoxyphenyl)-1,3-dithiad-
iphosphetane-2,4-disulfide) (0.44 g, 1.09 mmol) in toluene (3 mL)
was stirred and heated at reflux for 4 h. The reaction mixture was
washed by water (3 mL) and extracted with toluene (3 × 3 mL).
The combined extracts were dried over anhydrous Na2SO4 and
filtered, the solvent was removed in a vacuum, and the residue was
purified by FC (EtOAc:hexane, 1:10, Rf ) 0.30) to give 0.39 g of
6 (1.320 mmol, 67% yield) as a colorless liquid.1H NMR (CD3-
Cl): δ 4.37 (m, 1H), 3.67-3.71 (m, 5H), 2.12-2.32 (m, 4H), 0.85
(s, 9H), -0.05 (s, 6H). 13C NMR (CD3Cl): δ 81.5 (d, J ) 3.9 Hz),
65.4 (d, J ) 6.8 Hz), 52.4 (d, J ) 6.9 Hz), 29.2 (d, J ) 84.5 Hz),
25.7 (s), -0.1 (s), -5.4 (s), -5.6 (s). 31P NMR (CD3Cl): δ 113.43
(s). MS (CI) m/z 297.1 (M++1, 100.00). HRMS, M++1, Found:
297.1128. Calcd for C11H26O3PSSi, 297.1146.
Methyl 3,4-Dihydroxyl-butane 1,3-Cyclic Phosphonothioate
(7). A solution of 6 (143 mg, 0.48 mmol) in THF (8 mL) was treated
consecutively with acetic acid (83 µL, 1.45 mmol) and tetrabuty-
lammonium fluoride trihydrate (457 mg, 1.45 mmol) at room
1
ester. (7.97 g, 31.13 mmol, 44%). H NMR (CDCl3): δ 7.33-
7.26 (m, 5H), 4.57 (s, 2H), 4.34 (m, 1H), 3.76 (d, J ) 10.8 Hz,
3H), 3.56 (m, 2H), 2.23 (m, 1H), 2.09 (m, 1H), 1.96 (m, 1H), 1.85

Antagonists of Lysophosphatidic Acid Receptors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17 5313
temperature. After the solution was stirred for 18 h, the reaction
was complete (TLC). The solvent was then evaporated under
reduced pressure, and the crude product was purified by FC on a
short column (acetone:hexane, 3:2, Rf ) 0.45) to afford 61 mg of
(t, J ) 8.0 Hz, 2H), 2.10-2.20 (m, 2H), 2.02 (m, 2H), 1.58 (m,
2H), 1.22 (m, 24H), 0.86 (t, J ) 6.9 Hz, 3H). 13C NMR (CDCl3):
δ 174.0 (s), 66.1 (s), 65.5 (s), 34.3 (s), 33.4 (s), 31.9 (s), 29.7 (s),
29.5 (s), 29.4 (s), 29.3 (s), 29.2 (s), 26.9 (s), 14.1 (s). 31P NMR
(CDCl3): δ 94.32 (s). MS (CI) m/z 407.1 (M++1, 100.00). HRMS,
M++1, Found: 407.2397. Calcd for C20H40O4PS, 407.2409.
Diethyl 1- and 1,1-Difluoro-(3S)-4-hydroxybutylphosphonate
(10a and 10b). These compounds were prepared as previously
described.17
1
a colorless liquid (0.33 mmol, 69% yield). H NMR (CD3Cl): δ
4.30 (m, 1H), 3.55-3.76 (m, 5H), 2.92 (m, 1H), 2.02-2.31 (m,
5H). 13C NMR (CD3Cl): δ 82.2 (d, J ) 3.8 Hz), 64.5 (d, J ) 6.1
Hz), 52.5 (d, J ) 6.8 Hz), 29.5 (d, J ) 92.5 Hz), 25.4 (s). 31P
NMR (CD3Cl): δ 113.99 (s). MS (CI) m/z 183.0 (M++1, 100.00).
HRMS, M++1, Found: 183.0245. Calcd for C5H12O3PS, 183.0246.
Methyl 3-Hydroxyl-4-oleoyloxylbutane 1,3-Cyclic Phospho-
nothioate (8a). To a solution of alcohol 7 (14 mg, 0.077 mmol)
and oleic acid (24 mg, 0.133 mmol) in dry CH2Cl2 (2 mL) at room
temperature was added dropwise a solution of DCC (19 mg, 0.093
mmol) and DMAP (4 mg, 0.038 mmol) in dry CH2Cl2 (2 mL).
The solution was stirred at room temperature for 16 h and filtered,
the solvent was removed, and the residue was purified by FC (n-
hexanes-EtOAc 2:1, Rf ) 0.40) to afford 26 mg (0.059 mmol,
1-Fluoro-(3S),4-dihydroxybutylphosphonate (11a). The phos-
phonate 10a (46 mg, 0.189 mmol) in 5 mL flask was dried in vacuo.
Anhydrous TMS bromide (0.25 mL, 1.89 mmol) and CH2Cl2 (0.5
mL) were added into the flask. The solution was stirred at room
temperature for 5 h. TMS bromide and volatile products were
evaporated under high vacuum during 6 h. The residue was
dissolved in MeOH/H2O (95%, 1.0 mL) and stirred for 30 min at
room temperature, and then thoroughly dried to provide 33 mg of
1
an oily product (0.176 mmol, 93%). H NMR (CDCl3): δ 4.90
1
77%) of 8a as a colorless liquid. H NMR (CD3Cl): δ 5.29 (m,
(m, 1H), 3.85 (m, 1H), 3.50 (m, 2H), 2.05 (m, 2H), 2.18-2.03 (m,
2H). 13C NMR (CDCl3): δ 87.7 (m), 70.1 (d, J ) 8.3 Hz), 68.4 (d,
J ) 13.1 Hz), 67.5 (s), 66.6 (s), 35.3 (m). 19F NMR (CDCl3): δ
-207.39 (1F, m), -212.58 (1F, m). 31P NMR (CDCl3): δ 17.57
(d, J ) 76.10 Hz), 18.00 (d, J ) 75.1 Hz). MS (CI) m/z 188.0
(M++1, 79.05). HRMS, M+-H2O, Found: 171.0223. Calcd for
C4H9FO5P, 171.0223.
2H), 4.42 (m, 1H), 4.26 (m, 1H), 4.07 (m, 1H), 3.72 (d, J ) 12.0
Hz, 3H), 1.60-2.35 (m, 10H), 1.23-1.27 (m, 22H), 0.85 (t, J )
6.9 Hz, 3H). 13C NMR (CD3Cl): δ 173.5 (s), 130.0 (s), 129.7 (s),
78.7 (d, J ) 10.7 Hz), 65.4 (d, J ) 6.9 Hz), 52.6 (d, J ) 6.9 Hz),
34.0 (s), 33.4 (s), 31.9 (s), 29.7 (s), 29.7 (s), 29.5 (s), 29.3 (s), 29.1
(s), 29.1 (s), 27.2 (s), 27.1 (s), 25.7 (s), 24.8 (s), 22.7 (s), 19.1 (s),
17.9 (s), 14.1 (s). 31P NMR (CD3Cl): δ 112.96 (s). MS (CI) m/z
447.1 (M+, 100.00). HRMS, M+, Found: 447.2632. Calcd for
C23H43O4PS, 447.2648.
1-Fluoro-(3S)-hydroxyl-4-oleoyloxylbutane 1,3-Cyclic Phos-
phonate (13a). The phosphonate 11a (29 mg, 0.154 mmol) in 100
mL flask was dried in vacuo. Anhydrous DMF (40 mL) was added
into the flask, followed by addition of dicyclohexylcarbodiimide
(DCC, 1.0 M CH2Cl2 solution, 1.4 equiv., 0.216 mL). The solution
was stirred at room temperature for 16 h at which point water (0.2
mL) was added and stirred for additional 10 min, and then all
solvent was removed under high vacuum. The residue was dissolved
in anhydrous pyridine (2 mL), and oleyl chloride (0.084 mL, 1.4
equiv) was added. After stirred for 16 h at room temperature, the
solvent was removed under vacuum. The resulting residue was
purified by FC (CHCl3:CH3OH:H2O, 65:25:4, Rf ) 0.30). Further
purification was performed by open column on Sephedex LH-20
(CHCl3:CH3OH, 7:3) and afforded 43 mg of a colorless liquid
3-Hydroxyl-4-oleoyloxylbutane 1,3-Cyclic Phosphonothioate
(9a). A solution of 8a (18 mg, 0.04 mmol) in 3 mL of tert-
butylamine was refluxed for 48 h. The excess tert-butylamine was
removed by evaporation, and the resulting residue was purified by
FC (CH2Cl2:CH3OH:H2O, 8:1:0.05, Rf ) 0.14). Further purification
was performed by open column on Dowex 50WX8 100-200 (H+
form, CHCl3) and afforded 14 mg of a colorless liquid (0.03 mmol,
1
75% yield.) H NMR (CD3OD): δ 5.34 (m, 2H), 4.44 (m, 1H),
4.20 (dd, J ) 12.0, 3.2 Hz, 1H), 4.09 (dd, J ) 11.6, 6.0 Hz, 1H),
2.35 (t, J ) 8.0 Hz, 2H), 2.10-2.20 (m, 2H), 2.02 (m, 4H), 1.61
(m, 2H), 1.31 (m, 22H), 0.89 (t, J ) 6.9 Hz, 3H). 13C NMR (CD3-
OD): δ 173.2 (s), 128.9 (s), 128.8 (s), 76.0 (s), 65.6 (s), 33.0 (s),
31.1 (s), 28.9 (s), 28.8 (s), 28.6 (s), 28.5 (s), 28.3 (s), 28.2 (s), 26.1
(s), 24.0 (s), 21.8 (s), 12.5 (s). 31P NMR (CD3OD): δ 93.88 (s).
MS (CI) m/z 433.3 (M++1, 100.00). HRMS, M++1, Found:
433.2544. Calcd for C22H41O4PS, 433.2547.
1
(0.100 mmol, 65% yield). H NMR (CD3OD): δ 5.23 (m, 2H),
4.67 (m, 1H), 4.30 (m, 1H), 4.03 (m, 2H), 2.25 (m, 2H), 1.92 (m,
4H), 1.50 (m, 2H), 1.20 (m, 22H), 0.77 (m, 3H). 19F NMR (CD3-
OD): δ -198.86 (1F, m), -203.94 (1F, m). 31P NMR (CD3OD): δ
29.50 (d, J ) 72.9 Hz), 30.10 (d, J ) 75.1 Hz). MS HRMS
(MALDI), M++Na, Found: m/z 457.2537. Calcd for C22H39FO5-
PNa, 457.2490.
Methyl 3-Hydroxyl-4-palmitoyloxylbutane 1,3-Cyclic Phospho-
nothioate (8b). To a solution of alcohol 7 (22 mg, 0.12 mmol)
with palmatic acid (34 mg, 0.13 mmol) in dry CH2Cl2 (2 mL) at
room temperature was added dropwise a solution of DCC (30 mg,
0.15 mmol) and DMAP (7 mg, 0.06 mmol) in dry CH2Cl2 (2 mL).
The solution was stirred at room temperature for 16 h and filtered,
the solvent was removed, and the residue was purified by FC (n-
hexanes-EtOAc 5:1, Rf ) 0.21) to afford 41 mg (0.10 mmol, 73%)
1,1-Difluoro-(3S)-4-dihydroxybutylphosphonate (11b). The
phosphonate 10b (34 mg, 0.130 mmol) in 5 mL flask was dried in
vacuo. Anhydrous TMS bromide (0.23 mL, 1.65 mmol) and CH2-
Cl2 (0.2 mL) were added into the flask. The solution was stirred at
room temperature for 5 h. TMS bromide and volatile products were
evaporated under high vacuum during 6 h. The residue was
dissolved in MeOH/H2O (95%, 1.0 mL) and stirred for 30 min at
room temperature and then thoroughly dried to provide the oily
1
of 8b as a white solid. H NMR (CD3Cl): δ 4.53 (m, 1H), 4.28
(dd, J ) 12.4, 3.6 Hz, 1H), 4.09 (dd, J ) 11.6, 5.2 Hz, 1H), 3.73
(d, J ) 12.0 Hz, 3H), 2.10-2.20 (m, 2H), 2.02 (m, 2H), 1.58 (m,
2H), 1.23-1.27 (m, 24H), 0.85 (t, J ) 6.8 Hz, 3H). 13C NMR (CD3-
Cl): δ 173.5 (s), 78.9 (d, J ) 5.3 Hz), 65.5 (d, J ) 6.8 Hz), 52.7
(d, J ) 6.1 Hz), 34.1 (s), 31.9 (s), 29.8 (s), 29.7 (s), 29.6 (s), 29.4
(s), 29.3 (s), 29.2 (s), 29.1 (s), 28.9 (s), 26.4 (s), 24.8 (s), 24.7 (s),
14.1 (s). 31P NMR (CD3Cl): δ 112.98 (s). MS (CI) m/z 421.2
(M++1, 100.00). HRMS, M++1, Found: 421.2544. Calcd for
C22H41O4PS, 421.2564.
1
product (20 mg, 0.097 mmol, 75%). H NMR (CDCl3): δ 4.08
(m, 1H), 3.50 (m, 2H), 2.20 (m, 2H). 13C NMR (CDCl3): δ 65.4
(s), 65.1 (s), 36.7 (m). 19F NMR (CDCl3): δ -113.90 (2F, m). 31
P
NMR (CDCl3): δ 6.93 (t, J ) 245.0 Hz). MS (CI) m/z 207.0
(M++1, 68.00). HRMS, M++1, Found: 207.0245. Calcd for
C4H10F2O5P, 207.0234.
1,1-Difluoro-3(S)-hydroxyl-4-oleoyloxylbutane 1,3-Cyclic Phos-
phonate (13b). The same procedure was used as for the preparation
of 13a. The resulting residue was purified by FC (CHCl3:CH3OH:
H2O, 65:25:4, Rf ) 0.50). Further purification was performed by
open column on Sephadex LH-20 (CHCl3:CH3OH, 7:3) and to give
a colorless liquid(49% yield). 1H NMR (CD3OD): δ 5.35 (m, 2H),
4.38 (m, 1H), 2.55 (m, 2H), 2.35 (t, J ) 7.6 Hz, 2H), 2.02 (m,
4H), 1.61 (m, 2H), 1.29 (m, 22H), 0.90 (t, J ) 7.2 Hz, 3H). 13C
NMR (CD3OD): δ 173.8 (s), 129.7 (s), 129.6 (s), 68.7 (s), 65.7
(s), 37.0 (m), 33.6 (s), 31.9 (s), 29.7 (s), 29.6 (s), 29.5 (s), 29.4 (s),
29.3 (s), 29.2 (s), 29.1 (s), 29.0 (s), 26.9 (s), 24.7 (s), 22.6 (s), 13.3
3-Hydroxyl-4-palmitoyloxylbutane 1,3-Cyclic Phosphono-
thioate (9b). A solution of 8b (38 mg, 0.090 mmol) in 3 mL of
tert-butylamine was refluxed for 72 h. The excess tert-butylamine
was removed by evaporation, and the resulting residue was purified
on silica gel (CH2Cl2/CH3OH, 4:1, Rf ) 0.49). Further purification
was performed by open column on Dowex 50WX8 100-200 (H+
form, CHCl3) and afforded 23 mg of a colorless liquid (0.056 mmol,
62% yield). 1H NMR (CDCl3): δ 7.96 (br, 1H), 4.52 (m, 1H), 4.32
(dd, J ) 12.0, 3.2 Hz, 1H), 4.08 (dd, J ) 11.6, 6.0 Hz, 1H), 2.32

5314 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17
Xu et al.
(s). 19F NMR (CD3OD): δ -115.0 (2F, m). 31P NMR (CD3OD):
δ 12.97 (s). MS HRMS (MALDI), M++Na, Found: 475.2505.
Calcd for C22H39F2O5PNa, 475.2401.
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Water Mixture. All titrations were performed by using AR-15 pH
meter (Fisher Scientific) in solutions of 0.10 M NaCl under argon
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High Throughput Ca2+ Measurements. RH7777 cells stably
expressing either LPA1, LPA2, or LPA3 were plated on poly-D-
lysine-coated culture wells at a density of 50000 cells/well, and
cultured overnight. The culture medium was then replaced with
modified Krebs solution (120 mM NaCl, 5 mM KCl, 0.62 mM
MgSO4, 1.8 mM CaCl2, 10 mM HEPES, 6 mM glucose, pH 7.4),
and the cells were serum starved for 6 h. Cells were loaded with
Fura-2 AM for 35 min in modified Krebs medium. Changes in
intracellular Ca2+ concentration were monitored as described52 by
measuring the ratio of emitted light intensity at 520 nm in response
to excitation by 340 and 380 nm wavelength lights, respectively.
Each well was monitored for 80 s. 50 µL of the test compound
(3X stock solution in modified Krebs) was added automatically to
each well 15 s after the start of the measurement. Time courses
were recorded using the SoftMax Pro software (Molecular Devices,
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calculating the difference between maximum and baseline ratio
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Acknowledgment. We thank the Human Frontier Science
Program (RG0073-2000B) and the NIH (HL070231 and
NS29632) for support to G.D.P., and the NIH for CA921160
to G.T.
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