Structure activity relationships of lysophosphatidic acid: Conformationally restricted backbone mimetics

Article abstract of DOI:10.1021/jm970809v

Lysophosphatidic acid (LPA) has associated with it an intriguing cell biology that is thought to be mediated through its interaction with G- protein coupled receptor(s). In an effort to extend the structure-activity relationships of LPA, we have produced

Full text of DOI:10.1021/jm970809v

J . Med. Chem. 1999, 42, 963-970
963
Str u ctu r e-Activity Rela tion sh ip s of Lysop h osp h a tid ic Acid : Con for m a tion a lly
Restr icted Ba ck bon e Mim etics
Darrin W. Hopper,† Seamus P. Ragan,‡ Shelley B. Hooks,§ Kevin R. Lynch,‡,§ and Timothy L. Macdonald*,†
Department of Chemistry, University of Virginia, McCormick Road, Charlottesville, Virgonia 22901, and Departments of
Pharmacology and Biochemistry, University of Virginia, Health Science Center, Charlottesville, Virginia 22908
Received December 1, 1997
Lysophosphatidic acid (LPA) has associated with it an intriguing cell biology that is thought
to be mediated through its interaction with G-protein coupled receptor(s). In an effort to extend
the structure-activity relationships of LPA, we have produced a series of LPA analogues in
which the glycerol core in LPA was replaced with conformationally restricted aryl substructures.
The aryl substructures encompassed aminophenol, resorcinol, dihydroxy benzophenone, and
tocopherol systems. The benzophenone moiety was investigated both as a conformationally
restricting substructure for LPA and as a possible photoreactive alkylating agent for the LPA
receptor(s). All LPA analogues were evaluated for their potency and efficacy in mobilizing
calcium ions from internal stores in MDA MB-231 cells. Ten of the 14 analogues exhibited
activity in this assay at doses up to 5 µM; none of the compounds exhibited nonreceptor-mediated
lytic activity at this maximal concentration. The receptor response showed surprising tolerance
for manipulation in the backbone region of LPA, although none of the compounds were
equipotent to LPA. This tolerance for a variety of structures has given us new leads into the
realization of novel agonists and antagonists of the LPA receptor(s).
In tr od u ction
subunit, and we found that most of these analogues
exhibit significantly depressed activities.7 However, the
glycerol subunit can be replaced by the N-acyl system
without loss of either potency or efficacy5 and by N-acyl-
L-serine with only a slight loss in activity. Interestingly,
the N-acyl-D-serine-based analogue is significantly less
active than LPA.8
Lysophosphatidic acid (LPA) (Figure 1) has received
increasing attention due to the variety of biological
responses that it evokes, including platelet aggregation,
smooth-muscle contraction, cell morphology changes,
and mitogenic effects.1 There exists evidence suggesting
that LPA is the endogenous ligand for one or more
G-protein coupled receptors.2-4 As part of our continued
interest in the structure-activity relationships (SARs)
of LPA and related lipid phosphoric acid mediators, we
have been investigating the effects on receptor-mediated
responses of alterations in the three substructural
domains of LPA. The substructural domains of LPAs
the “LPA pharmacophore”smay be postulated to consist
of the phosphate “head” group, a “linker” region il-
lustrated by glycerol in LPA, and a lipophilic “tail”
illustrated by the fatty acyl chain of LPA (Figure 1). The
studies reported here and our previous studies have
used calcium mobilization and/or inhibition of adenylyl
cyclase in cultured MDA MB-231 cells as a measure of
receptor-mediated responses.
Our previous SAR studies have shown that the
phosphate group is critical for activity, since all of the
phosphate surrogates examined to date have resulted
in sharply diminished activities in calcium mobilization
and inhibition of adenylyl cyclase5 (unpublished data).
In contrast, our studies of the lipophilic “tail” have found
that while oleic acid exhibits the greatest potency,
considerable variation in the fatty acyl chain length and
level of unsaturation can be tolerated, reminiscent of
PAF (unpublished data).6 We have also investigated a
family of 2-5 carbon analogues of the glycerol “linker”
To expand our knowledge concerning the SAR profile
of the LPA receptor we wanted to investigate further
the extent of structural tolerance permitted in the
backbone region. We report now the replacement of the
glycerol backbone of LPA with a variety of aromatic
moieties (Figure 2). Further impetus for this synthetic
effort was the hope that we would identify structures
that might have antagonistic activity. Moreover, we
wished to identify photoreactive alkylating agents for
use in labeling putative LPA receptors. In our previous
studies of LPA SAR, we determined that the hydroxyl
at the 2 position of glycerol is not necessary for agonist
activity; however, this functionality may play an im-
portant role in solubility.5 The most successful com-
pound was realized by the replacement of the glycerol
backbone with ethanolamine. The proton of the amide
functionality in the N-acyl ethanolamine analogue
might replace a requirement that the hydroxyl proton
on the glycerol backbone fulfills, since both protons are
five bonds away from the phosphorus atom (Figure 3).
We also determined that fatty acid chains with unsat-
uration are more active than saturated fatty acid chains
(unpublished data). Although the activity differential
between unsaturated and saturated fatty acid groups
could be a consequence of binding to the receptor(s), we
believe that an important contribution to this activity
differential is the enhanced solubility in aqueous solu-
tions of the unsaturated fatty acid systems relative to
† Department of Chemistry.
‡ Department of Pharmacology.
§ Department of Biochemistry.
10.1021/jm970809v CCC: $18.00 © 1999 American Chemical Society
Published on Web 03/02/1999

964 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 6
Hopper et al.
F igu r e 1.
F igu r e 2.
produced an aryl diazirine alkylating agent that alkyl-
ated a putative LPA receptor upon photoactivation. To
overcome the high reactivity and concomitant low
selectivity of the carbene species generated by diazirine
photodecomposition, we replaced the glycerol in the
linker region of LPA with a benzophenone moiety. The
benzophenone moiety has been reported to possess high
selectivity for the photolabeling of proteins.9 We hoped
that these benzophenone-based systems might prove
useful as photoactivated probes for the putative LPA
receptor(s).
Ch em istr y
Four classes of conformationally restricted LPA ana-
logues were synthesized. Each class possessed a differ-
ent aromatic backbone in place of the traditional
glycerol backbone in phospholipids. The length of the
fatty acyl chains were chosen to approximate the overall
length of 1-oleoyl-LPA. The synthesis of each class is
described below.
The resorcinol and aminophenol backboned mimetics
were synthesized by similar methods (Scheme 1). O- or
N-acylation was accomplished by condensation of the
appropriate acid chloride with the aryl derivatives,
followed by phosphorylation of the remaining hydroxyl
with dibenzyl diisopropylphosphoramidite. The benzyl
protection groups on the phosphate triester were re-
moved by hydrogenolysis to yield the desired lipid
phosphate.
F igu r e 3.
their saturated counterparts, thereby enabling greater
effective concentrations.
We also desired a mimetic that would be capable of
alkylating the receptor. Previously, van der Bend et al.2

SARs of Lysophosphatidic Acid
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 6 965
Sch em e 1
Sch em e 2
Ta ble 1. Relative Potencies of the 10 Active LPA Mimetics
The synthesis of the benzophenone class was achieved
by the monoacylation of 4,4′-dihydroxybenzophenone
with the appropriate acid chloride, followed by phos-
phorylation of the remaining hydroxyl with di-tert-butyl
diisopropylphosphoramidite. The removal of the protect-
ing groups on the phosphate triester was accomplished
by acid hydrolysis (Scheme 2).
The last class is the least developed of the four.
R-Tocophorol was phosphorylated and deprotected as in
Scheme 2. This was done to explore the further restric-
tion of the backbone region beyond that of the previous
three classes.
Synthesizeda
compd
EC50s (µM) MDA MB 231 cells
7
2
5
8
3
6
0.31 ( 0.11
0.46 ( 0.13
0.46 ( 0.05
0.85 ( 0.29*
1.56 ( 0.82*
1.83 ( 0.64
1.99 ( 0.65*
4.71 ( 0.68*
>500*
13
12
11
10
15
LPA
>500*
0.003
0.005
Resu lts a n d Discu ssion
a
The EC50 values with an asterisk denote those that did not
A series of LPA analogues 1-14, in which the glycerol
backbone of LPA was replaced by aminophenol, resor-
cinol, benzophenone, and R-tocopherol aromatic groups,
were assayed for their ability to mobilize intracellular
calcium in MDA MB-231 cells. Each of the aminophenol-
and resorcinol-based analogues were acylated with
palmitoyl or palmitoleoyl fatty acid chains to ap-
proximate the length of oleoyl-LPA, which is the most
potent LPA derivative and our standard for assay. Table
1 summarizes the SAR data for calcium ion mobilization
in MDA MB-231 cells, while the dose-response curves
are shown in Figure 4.
The o-aminophenol analogues 1 and 4 were antici-
pated to be the most effective compounds, due to their
structural similarity to N-fatty acyl ethanolamide phos-
phate 15 (Figure 3), which is almost equipotent to LPA
(EC50 ) 3 nM). A simple analysis suggests that these
reach a maximal response at the highest concentration used.
o-aminophenol derivatives 1 and 4 could be viewed as
“conformationally restricted” analogues of ethanolamide
phosphate 15. However, these ortho-substituted ana-
logues (1 and 4) proved to be completely inactive at the
highest concentration tested (5 µM) (Table 1). The
inactivity of 1 and 4 could reflect a structurally dictated
and incorrect “syn” relationship of the 2-amido phos-
phate system, as compared to an “anti” relationship
possibly required for activity. It is also possible that a
hydrogen bond that can be formed between the amide
functionality and the phosphate headgroup (Figure 3)
serves to further “lock” the conformation of these
analogues, thereby inhibiting minor structural adjust-
ments of the system necessary to adopt the “active”
conformation. In addition, such a H-bonding would

966 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 6
Hopper et al.
benzophenone mimic 9 exhibited no activity, which we
attribute to its having an insufficiently long fatty acid
chain (hexyl). Although 13 was the most active benzo-
phenone mimic, it was considerably less potent than
LPA (EC50s: LPA ) 5 nM, 13 ) 1.99 µM). However, it
is possible that the diminished potency of these ben-
zophenone species will be counteracted by the increased
efficiency of protein cross-linking by the benzophenone
subunit.
The benzophenone-based analogues show that the
putative LPA receptor(s) have a surprisingly large
tolerance for substitution in the linker region. This led
us to further explore aromatic linker mimics. Phospho-
rylated R-tocopherol, the fat-soluble vitamin, has the
structural elements identified in these studies for activ-
ity and we hypothesized that this species might function
as a LPA mimetic. However, the phosphorylated form
of R-tocopherol 14 was not an agonist for calcium
mobilization in MDA MB 231 cells at concentrations up
to 5 µM.
The increased rigidity that these aromatic substruc-
tures confer on these analogues might result in com-
pounds that bind to the receptor but do not enable the
conformational alteration needed to express activity.
Therefore, the LPA analogues that showed no detectable
agonist activity (1, 4, 9, 14) were tested to see if they
exhibited antagonistic activity. To test for antagonist
activity, the effects of the analogues were monitored on
the Ca2+ response in MDA MB-231 cells to LPA. In
practice, our antagonist assay examined the Ca2+
response to LPA at 1 and 10 nM individually. These
results were compared to the Ca2+ response of samples
that contained (1 or 10 nM) LPA and analogues 1, 4, 9,
or 14 at 1 µM. None of these analogues (1, 4, 9, or 14)
proved to affect the response of LPA at either concen-
tration.
F igu r e 4. Dose-response curves for calcium mobilization in
MDA MB-231 cells. Calcium mobilization was measured as a
function of INDO-1AM fluorescence enhancement and pre-
sented as a percent of the total available calcium defined by
cellular lysis with digitonin. The values for analogues were
normalized to the maximal LPA response which is typically
30% of the total amount of calcium released by digitonin. All
points used to calculate the EC50 values were done in triplicate
and were curve-fitted with GraphPAD (GraphPAD software,
San Diego, CA). The EC50 values for these curves are presented
in Table 1.
depress the charge density at the phosphate headgroup
along with depleting the compound of a possible hydro-
gen accepting site. Previous studies by other laborato-
ries8 and our own laboratory (unpublished data) showed
that the lack of a dianionic phosphate destroys agonistic
activity. We believe that the depressed activity of the
ortho-substituted aminophenol derivatives relative to
the aminophenol derivatives is not simply a consequence
of the increased steric bulk of the “linker unit”. This
belief is based on the significant activity of the meta-
substituted and benzophenone-based analogues dis-
cussed below.
Interestingly, the most active of the aminophenol-
based compounds were the meta-substituted derivatives
2, 5, and 7 (Table 1). These compounds adopted a
conformation that more closely mimics an “anti” rela-
tionship between the fatty acid moiety and the phos-
phate as viewed by molecular modeling of these ana-
logues. In addition, compounds 2, 5, and 7 have a three-
carbon backbone separating the phosphate and amide
or ester functionalities analogous to LPA, but they lack
the 2-hydroxyl moiety. The para-substituted LPA ana-
logue 3 was less active than the meta-derivatives 2, 5,
and 7. This observation parallels our previous finding
that as the distance between the phosphate and ester
or amide functionality is increased in aliphatic backbone
compounds,5 activity is decreased.
A third set of lipid phosphate species incorporated a
benzophenone moiety as a potential photoaffinity probe.
The benzophenone moiety has the ability to be activated
to its excited state at wavelengths not associated with
protein damage (>320 nm) and is highly efficient in its
ability to insert covalently into reactive C-H bonds.9
Moreover, the benzophenone moiety can be manipulated
in ambient light during experimental conditions. The
myristylated benzophenone analogue 13 was the most
active (Table 1), and our progress in applying this
reagent as a photoaffinity probe in labeling experiments
will be reported in due course. As seen in Table 1, the
ability of the benzophenone analogues to mobilize
calcium decreases sharply as the numbers of methylene
subunits in the fatty acid chain is reduced. Only
The knowledge gained from this SAR study illustrated
considerable tolerance of the LPA receptor(s) for struc-
tural alterations in the backbone region. Our studies
significantly enhance our knowledge concerning the
structural requirements for activity at the LPA receptor
presented in previously reported studies. Although we
did not produce an agonist with equal activity to LPA,
these novel LPA analogues provide a variety of new lead
structures that can used for further development of
LPA-based agonists.
Exp er im en ta l Section
All melting points were taken on a Thomas-Hoover UNI-
MELT melting point apparatus and are uncorrected. All
solvents were filtered through appropriate desiccant under
nitrogen immediately prior to use. All nuclear magnetic
resonance spectra were obtained with a General Electric
QE300 spectrometer at 300 MHz, and chemical shifts are
reported in ppm. Elemental analysis were performed either
by Atlantic Microlab. Inc., Norcross, GA, or at the University
of Virginia on a Perkin-Elmer PE 2400 C, H, N analyzer. Thin-
layer chromatography was performed on Merck silica gel 60
F-254 precoated plates, and visualization was effected with
phosphomolybdic acid in ethanol. The thin-layer chromatog-
raphy Rf values reported were obtained using the chromato-
graphic solvent system described for large scale chromato-
graphic purification using 2-hydroxyethyl-(9Z)-octadec-9-
enoate as an internal standard. The Rf values for 2-hydroxy-
ethyl-(9Z)-octadec-9-enoate obtained under the following con-
ditions were as follows: 20% ethyl acetate in hexanes, Rf )
0.14; 30% ethyl acetate in hexanes, Rf ) 0.29; 5% acetone in

SARs of Lysophosphatidic Acid
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 6 967
chloroform, Rf ) 0.45; 15% acetone in chloroform, Rf ) 0.55.
Column chromatography was performed on E. Merck silica 60
(230-400 mesh). Radial chromatography was executed on a
Chromatotron 7924 from Harrison Research System.
0.18 mmol) was dissolved in 2 mL of a 1:1 mixture of THF/
cyclohexene. To this solution was added 10% Pd/C, and the
resulting mixture was stirred for 4 h at 78 °C and under a
nitrogen atmosphere. The reaction mixture was brought to
room temperature and then filtered through a layer of Celite
using a 1:1 methanol/chloroform mixture. The organic layer
was removed under reduced pressure to give the desired lipid
phosphate analogue.
Gen er a l P r oced u r e H: Dep r otection of ter t-Bu tyl
P r otected P h osp h otr iester Sp ecies. 4-(4-((Di(tert-butoxy)-
phosphoryl)oxy)benzoyl)phenyl hexanoate (32) (0.11 g, 0.17
mmol) was dissolved in 1.5 mL of ethyl acetate to which 2
drops of concentrated HCl was added. The reaction mixture
was stirred for 6 h at room temperature followed by the
removal of the solvents under reduced pressure to give the
desired lipid phosphate analogue.
Gen er a l P r oced u r e A: Acyla tion of Dih yd r oxyben zo-
p h en on es. Hexanoyl chloride (0.36 mL, 2.6 mmol) was added
to a solution of 4-4′-dihydroxybenzophenone (1.00 g, 5.2 mmol)
and pyridine (0.21 mL, 2.6 mmol) in 50 mL of THF and stirred
at room-temperature overnight. The reaction mixture was
diluted with ethyl acetate (75 mL) and extracted with satu-
rated aqueous NH4Cl (3 × 75 mL). The organic layer was dried
over Na2SO4. The solvent was removed under reduced pres-
sure, and the product was purified by flash chromatography
using 5% acetone in chloroform.
Gen er a l P r oced u r e B: Acyla tion of th e Am in op h en ols.
Pamitoyl chloride (1.26 g, 4.6 mmol) was added to a solution
of 2-aminophenol (1.00 g, 9.16 mmol) in 100 mL of THF and
stirred at room temperature overnight. The reaction mixture
was diluted with ethyl acetate (150 mL) and extracted with
saturated aqueous NH4Cl (3 × 100 mL). The organic layer was
dried over Na2SO4. The solvent was removed under reduced
pressure, and the product was purified by flash chromatog-
raphy using 15% acetone in chloroform.
N-1-(2-Hyd r oxyp h en yl) Hexa d eca n a m id e (16). This
aminophenol analogue was prepared as described in procedure
B (80% yield) and purified by flash chromatography using 15%
acetone in chloroform to afford a white solid: mp 79-80 °C;
1
Rf ) 0.25; H NMR (300 MHz, CDCl3) δ 8.84 (s, 1H), 7.42 (s,
1H), 7.26-6.83 (m, 4H), 2.45 (t, J ) 7.32 Hz, 2H), 1.72 (q, J )
6.93 Hz, 2H), 1.26 (s, 24H), 0.88 (t, J ) 6.93 Hz, 3H).
Gen er a l P r oced u r e C: Acyla tion of Resor cin ol. Pami-
toyl chloride (1.25 g, 4.5 mmol) was added to a solution of
resorcinol (1.00 g, 9.1 mmol) and pyridine (0.36 mL, 4.5 mmol)
in 100 mL of THF and stirred overnight at room temperature.
The reaction mixture was diluted with ethyl acetate (150 mL)
and extracted with saturated aqueous NH4Cl (3 × 100 mL).
The organic layer was dried over Na2SO4. The solvent was
removed under reduced pressure, and the product was purified
by flash chromatography using 5% acetone in chloroform.
Gen er a l P r oced u r e D: Hyd r oxyl P h osp h or yla tion of
th e Am in op h en ol An a logu es. Dibenzyl diisopropylphos-
phoramidite (0.31 mL, 0.93 mmol) was added to a solution of
N-1-(2-hydroxyphenyl)hexadecanamide (16) (200 mg, 0.57
mmol) and tetrazole (65 mg, 0.93 mmol) in 50 mL of 1:1 THF/
CH2Cl2. The reaction was stirred at room temperature over-
night, and 30% aqueous H2O2 (0.2 mL) was added and stirred
for an additional 2 h. The reaction was then brought to 0 °C,
and excess H2O2 was quenched with saturated aqueous sodium
metabisulfite. The resulting reaction mixture was diluted with
ethyl acetate (50 mL) and extracted with saturated aqueous
sodium metabisulfite (2 × 50 mL). The organic layer was dried
over Na2SO4. The solvent was removed under reduced pres-
sure, and the product was purified by radial chromatography
using 20% ethyl acetate in hexanes.
Gen er a l P r oced u r e E: Hyd r oxyl P h osp h or yla tion of
t h e Ben zop h en on e An a logu es. Di-tert-butyl diisopropyl-
phosphoramidite (0.31 g, 1.13 mmol) was added to a solution
of 4-(4-hydroxybenzoyl)phenyl octanoate (33) (0.24 g, 0.70
mmol) and tetrazole (0.80 g, 1.13 mmol) in 50 mL of 1:1 THF/
CH2Cl2. The reaction was stirred at room temperature over-
night, and 30% aqueous H2O2 (0.2 mL) was added and stirred
for an additional 2 h. The reaction was then brought to 0 °C,
and excess H2O2 was quenched with saturated aqueous sodium
metabisulfite. The resulting reaction mixture was diluted with
ethyl acetate (50 mL) and extracted with saturated aqueous
sodium metabisulfite (2 × 50 mL). The organic layer was dried
over Na2SO4. The solvent was removed under reduced pres-
sure, and the product was purified by radial chromatography
using 20% ethyl acetate in hexanes.
N-1-(3-Hyd r oxyp h en yl) Hexa d eca n a m id e (17). This
aminophenol analogue was prepared as described in procedure
B (82% yield) and purified by flash chromatography using 15%
acetone in chloroform to afford a white solid: mp 112-114 °C;
1
Rf ) 0.25; H NMR (300 MHz, CDCl3) δ 7.75-6.61 (m, 4H),
2.37 (t, J ) 6.93 Hz, 2H), 1.73 (q, J ) 6.93 Hz), 1.26 (s, 24H),
0.88 (t, J ) 6.93 Hz, 3H).
N-1-(4-Hyd r oxyp h en yl) Hexa d eca n a m id e (18). This
aminophenol analogue was prepared as described in procedure
B (84% yield) and purified by flash chromatography using 15%
acetone in chloroform to afford a white solid: mp 133 °C (dec);
Rf ) 0.25; 1H NMR (300 MHz, CD3OD) δ 7.34 (d, J ) 8.86,
2H), 6.60 (d, J ) 8.86, 2H), 2.15 (t, J ) 7.7 Hz, 2H), 1.45 (q, J
) 6.94 Hz, 2H), 1.09 (s, 24H), 0.68 (t, J ) 6.93 Hz, 3H).
3-Hyd r oxyp h en yl P a lm ita te (19). This resorcinol ana-
logue was prepared as described in procedure C (36% yield)
and purified by flash chromatography using 5% acetone in
chloroform to afford a white solid: mp 66-68 °C; Rf ) 0.25;
1H NMR (300 MHz, CDCl3) δ 7.20-6.32 (m, 4H), 2.54 (t, J )
7.7 Hz, 2H), 1.75 (q, J ) 7.7 Hz, 2H), 1.26 (s, 24H), 0.89 (t, J
) 6.94 Hz, 3H).
N-1-(2-H yd r oxyp h en yl) (Z)-9-H exa d ecen a m id e (20).
This aminophenol analogue was prepared as described in
procedure B (70% yield) and purified by flash chromatography
using 15% acetone in chloroform to afford a white solid: mp
36-38 °C; Rf ) 0.25; 1H NMR (300 MHz, CDCl3) δ 8.84 (s,
1H), 7.42 (s, 1H), 7.12-6.83 (m, 4H), 5.35 (m, 2H), 2.43 (t, J )
7.32 Hz, 2H), 2.02 (m, 4H), 1.72 (q, J ) 7.31 Hz, 2H), 1.32 (m,
16H), 0.88 (t, J ) 7.32 Hz, 3H).
N-1-(3-Hyd r oxyp h en yl) (Z)-9-Hexa d ecen a m id e (21).
This aminophenol analogue was prepared as described in
procedure B (67% yield) and purified by flash chromatography
using 15% acetone in chloroform to afford a white solid: mp
62-64 °C; Rf ) 0.25; 1H NMR (300 MHz, CDCl3) δ 7.12-6.54
(m, 4H), 5.35 (m, 2H), 2.34 (t, J ) 7.32 Hz, 2H), 2.02 (m, 4H),
1.72 (q, J ) 7.31 Hz, 2H), 1.32 (m, 16H), 0.88 (t, J ) 7.32 Hz,
3H).
N-1-(4-Hyd r oxyp h en yl) (Z)-9-Hexa d ecen a m id e (22).
This aminophenol analogue was prepared as described in
procedure B (84% yield) and purified by flash chromatography
using 15% acetone in chloroform to afford a white solid: mp
Gen er al P r ocedu r e F: Depr otection of Lipid P h osph o-
tr iester s w ith Sa tu r a ted F a tty Acid Ch a in s. Dibenzyl (2-
(palmitoylamino)phenyl) phosphate (24) (0.14 g, 0.16 mmol)
was dissolved in 2 mL of THF. To this solution was added 10%
Pd/C (catalytic), and the resulting mixture was placed under
H2 atmosphere for 2 h. The reaction mixture was then filtered
through a layer of Celite using a 1:1 solution of methanol and
chloroform. The organic layer was removed under reduced
pressure to give the desired lipid phosphate analogue.
Gen er al P r ocedu r e G: Depr otection of Lipid P h osph o-
tr iester s w ith Un sa tu r a ted F a tty Acid Ch a in s. Dibenzyl
(2-((Z)-9-hexadecenoylamino)phenyl) phosphate (28) (0.11 g,
1
101-103 °C; Rf ) 0.25; H NMR (300 MHz, CDCl3) δ 7.27 (d,
J ) 8.45 Hz, 2H), 6.75 (d, J ) 8.60 Hz, 2H), 5.35 (m, 2H), 2.33
(t, J ) 7.32 Hz, 2H), 2.02 (m, 4H), 1.72 (q, J ) 7.31 Hz, 2H),
1.32 (m, 16H), 0.88 (t, J ) 7.32 Hz, 3H).
3-Hyd r oxyp h en yl (Z)-9-Hexa d ecen oa te (23). This re-
sorcinol analogue was prepared as described in procedure C
(38% yield) and purified by flash chromatography using 5%
1
acetone in chloroform to afford a clear oil: Rf ) 0.5; H NMR
(300 MHz, CDCl3) δ 7.20-6.32 (m, 4H), 5.36 (m, 2H), 2.54 (t,

968 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 6
Hopper et al.
J ) 7.7 Hz, 2H), 2.03 (m, 4H), 1.75 (q, J ) 7.7 Hz, 2H), 1.32
(m, 16H), 0.89 (t, J ) 6.94 Hz, 3H).
Hz, 4H), 2.53 (t, J ) 7.7 Hz, 2H), 1.74 (q, J ) 7.31 Hz, 2H),
1.28 (s, 24H), 0.89 (t, J ) 6.55 Hz, 3H).
Diben zyl (2-((Z)-9-Hexa d ecen oyla m in o)p h en yl) P h os-
p h a te (28). This aminophenol phosphate analogue was pre-
pared as described in procedure D (75% yield) and purified by
radial chromatography using 30% ethyl acetate in hexanes to
afford a clear oil: Rf ) 0.26; 1H NMR (300 MHz, CDCl3) δ
7.33-6.94 (m, 4H), 7.31 (s, 10H), 5.35 (m, 2H), 5.1 (d, J ) 8.85
Hz, 4H), 2.24 (t, J ) 7.71 Hz, 2H), 2.0 (m, 4H), 1.66 (q, J )
7.31 Hz, 2H), 1.29 (m, 24H), 0.88 (t, J ) 6.94 Hz, 3H).
Diben zyl (3-((Z)-9-Hexa d ecen oyla m in o)p h en yl) P h os-
p h a te (29). This aminophenol phosphate analogue was pre-
pared as described in procedure D (78% yield) and purified by
radial chromatography using 30% ethyl acetate in hexanes to
afford a clear oil: Rf ) 0.26; 1H NMR (300 MHz, CDCl3) δ
7.44-6.84 (m, 4H), 7.31 (s, 10H), 5.35 (m, 2H), 5.12 (d, J )
8.47 Hz, 4H), 2.31 (t, J ) 7.71 Hz, 2H), 2.0 (m, 4H), 1.68 (q, J
) 7.31 Hz, 2H), 1.31 (m, 24H), 0.88 (t, J ) 6.94 Hz, 3H).
Diben zyl (4-((Z)-9-Hexa d ecen oyla m in o)p h en yl) P h os-
p h a te (30). This aminophenol phosphate analogue was pre-
pared as described in procedure D (79% yield) and purified by
radial chromatography using 30% ethyl acetate in hexanes to
afford a clear oil: Rf ) 0.26; 1H NMR (300 MHz, CDCl3) δ 7.41
(d, J ) 8.85 Hz, 2H), 7.33 (s, 10H), 7.0 (d, J ) 8.85 Hz, 2H),
5.35 (m, 2H), 5.1 (d, J ) 8.47 Hz, 4H), 2.33 (t, J ) 7.7 Hz,
2H), 2.0 (m, 4H), 1.69 (m, 2H), 1.27 (m, 24H), 0.88 (t, J ) 6.69
Hz, 3H).
4-(4-Hyd r oxyben zoyl) P h en yl Hexa n oa te (32). This
benzophenone analogue was prepared as described in proce-
dure A (30% yield) and purified by flash chromatography using
5% acetone in chloroform to afford a white solid: mp 85-87
1
°C; Rf ) 0.55; H NMR (300 MHz, CDCl3) δ 7.79 (d, J ) 8.48
Hz, 2H), 7.73 (d, J ) 8.85 Hz, 2H), 7.20 (d, J ) 8.86 Hz, 2H),
6.90 (d, J ) 8.47 Hz, 2H), 2.60 (t, J ) 7.7 Hz, 2H), 1.78 (q, J
) 7.32 Hz, 2H), 1.38 (m, 4H), 0.93 (t, J ) 7.31 Hz, 3H).
4-(4-Hyd r oxyben zoyl) P h en yl Octa n oa te (33). This ben-
zophenone analogue was prepared as described in procedure
A (35% yield) and purified by flash chromatography using 5%
acetone in chloroform to afford a white solid: mp 86-87 °C;
1
Rf ) 0.55; H NMR (300 MHz, CDCl3) δ 7.79 (d, J ) 8.48 Hz,
2H), 7.73 (d, J ) 8.85 Hz, 2H), 7.20 (d, J ) 8.86 Hz, 2H), 6.90
(d, J ) 8.47 Hz, 2H), 2.60 (t, J ) 7.7 Hz, 2H), 1.78 (q, J ) 7.32
Hz, 2H), 1.38 (m, 4H), 0.93 (t, J ) 7.31 Hz, 3H).
4-(4-Hyd r oxyben zoyl) P h en yl Deca n oa te (34). This
benzophenone analogue was prepared as described in proce-
dure A (33% yield) and purified by flash chromatography using
5% acetone in chloroform to afford a white solid: mp 87-88
1
°C; Rf ) 0.55; H NMR (300 MHz, CDCl3) δ 7.79 (d, J ) 8.86
Hz, 2H), 7.73 (d, J ) 8.85 Hz, 2H), 7.20 (d, J ) 8.86 Hz, 2H),
6.90 (d, J ) 8.86 Hz, 2H), 2.60 (t, J ) 7.7 Hz, 2H), 1.78 (q, J
) 7.32 Hz, 2H), 1.27 (m, 12H), 0.88 (t, J ) 7.31 Hz, 3H).
4-(4-Hyd r oxyben zoyl) P h en yl La u r a te (35). This ben-
zophenone analogue was prepared as described in procedure
A (33% yield) and purified by flash chromatography using 5%
acetone in chloroform to afford a white solid: mp 89-91 °C;
3-((Di)ben zyloxy)p h osp h or yl)oxy) P h en yl (Z)-9-Hexa -
d ecen oa te (31). This resorcinol phosphate analogue was
prepared as described in procedure D (65% yield) and purified
by radial chromatography using 20% ethyl acetate in hexanes
1
Rf ) 0.55; H NMR (300 MHz, CDCl3) δ 7.79 (d, J ) 8.48 Hz,
1
to afford a clear oil: Rf ) 0.32; H NMR (300 MHz, CDCl3) δ
2H), 7.73 (d, J ) 8.47 Hz, 2H), 7.20 (d, J ) 8.86 Hz, 2H), 6.90
(d, J ) 8.86 Hz, 2H), 2.60 (t, J ) 7.7 Hz, 2H), 1.78 (q, J ) 7.32
Hz, 2H), 1.27 (m, 16H), 0.88 (t, J ) 7.31 Hz, 3H).
7.32 (s, 10H), 7.30-6.91 (m, 4H), 5.37 (m, 2H), 5.12 (d, J )
8.47 Hz, 4H), 2.53 (t, J ) 7.7 Hz, 2H), 2.05 (m, 2H), 1.75 (q, J
) 7.31 Hz, 2H), 1.32 (m, 24H), 0.89 (t, J ) 6.93 Hz, 3H).
4-(4-((Di(ter t-b u t oxy)p h osp h or yl)oxy)b en zoyl)p h en -
yl Hexa n oa te (37). This benzophenone phosphate analogue
was prepared as described in procedure E (76% yield) and
purified by radial chromatography using 20% ethyl acetate in
4-(4-Hyd r oxyben zoyl) P h en yl Myr ista te (36). This ben-
zophenone analogue was prepared as described in procedure
A (36% yield) and purified by flash chromatography using 5%
acetone in chloroform to afford a white solid: mp 94-96 °C;
1
Rf ) 0.55; H NMR (300 MHz, CDCl3) δ 7.79 (d, J ) 8.48 Hz,
1
hexanes to afford a clear oil: Rf ) 0.55; H NMR (300 MHz,
2H), 7.73 (d, J ) 8.86 Hz, 2H), 7.20 (d, J ) 8.47 Hz, 2H), 6.90
(d, J ) 8.86 Hz, 2H), 2.60 (t, J ) 7.7 Hz, 2H), 1.78 (q, J ) 7.7
Hz, 2H), 1.27 (m, 20H), 0.88 (t, J ) 6.93 Hz, 3H).
CDCl3) δ 7.81-7.77 (m, 4H), 7.29 (d, J ) 8.86 Hz, 2H), 7.18
(d, J ) 8.47 Hz, 2H), 2.57 (t, J ) 7.7 Hz, 2H), 1.73 (q, J ) 7.7
Hz, 2H), 1.52 (s, 18H), 1.38 (m, 4H), 0.91 (t, J ) 7.31 Hz, 3H).
4-(4-((Di(ter t-b u t oxy)p h osp h or yl)oxy)b en zoyl)p h en -
yl Octa n oa te (38). This benzophenone phosphate analogue
was prepared as described in procedure E (78% yield) and
purified by radial chromatography using 20% ethyl acetate in
Diben zyl (2-(P a lm itoyla m in o)p h en yl) P h osp h a te (24).
This aminophenol phosphate analogue was prepared as de-
scribed in procedure D (79% yield) and purified by radial
chromatography using 30% ethyl acetate in hexanes to afford
1
a white solid: mp 48-50 °C; Rf ) 0.26; H NMR (300 MHz,
1
hexanes to afford a clear oil: Rf ) 0.55; H NMR (300 MHz,
CDCl3) δ 7.33-6.94 (m, 4H), 7.31 (s, 10H), 5.1 (d, J ) 8.85
Hz, 4H), 2.24 (t, J ) 7.71 Hz, 2H), 1.66 (q, J ) 7.31 Hz, 2H),
1.27 (s, 24H), 0.88 (t, J ) 6.94 Hz, 3H).
CDCl3) δ 7.81-7.74 (m, 4H), 7.31 (d, J ) 8.09 Hz, 2H), 7.19
(d, J ) 8.45 Hz, 2H), 2.57 (t, J ) 7.321 Hz, 2H), 1.76 (m, 2H),
1.53 (s, 18H), 1.31 (m, 8H), 0.89 (t, J ) 6.16 Hz, 3H).
Diben zyl (3-(P a lm itoyla m in o)p h en yl) P h osp h a te (25).
This aminophenol phosphate analogue was prepared as de-
scribed in procedure D (88% yield) and purified by radial
chromatography using 30% ethyl acetate in hexanes to afford
4-(4-((Di(ter t-b u t oxy)p h osp h or yl)oxy)b en zoyl)p h en -
yl Deca n oa te (39). This benzophenone phosphate analogue
was prepared as described in procedure E (70% yield) and
purified by radial chromatography using 20% ethyl acetate in
1
1
a white solid: mp 46-48 °C; Rf ) 0.26; H NMR (300 MHz,
hexanes to afford a white solid: mp 43-44 °C; Rf ) 0.55; H
CDCl3) δ 7.36-6.82 (m, 4H), 7.30 (s, 10H), 5.1 (d, J ) 8.47
Hz, 4H), 2.3 (t, J ) 7.31 Hz, 2H), 1.66 (m, 2H), 1.27 (s, 24H),
0.88 (t, J ) 6.74 Hz, 3H).
NMR (300 MHz, CDCl3) δ 7.81-7.77 (m, 4H), 7.29 (d, J ) 8.09
Hz, 2H), 7.19 (d, J ) 8.47 Hz, 2H), 2.56 (t, J ) 7.7 Hz, 2H),
1.71 (m, 2H), 1.51 (s, 18H), 1.27 (m, 12H), 0.85 (t, J ) 7.31
Hz, 3H).
Diben zyl (4-(P a lm itoyla m in o)p h en yl) P h osp h a te (26).
This aminophenol phosphate analogue was prepared as de-
scribed in procedure D (61% yield) and purified by radial
chromatography using 30% ethyl acetate in hexanes to afford
4-(4-((Di(ter t-b u t oxy)p h osp h or yl)oxy)b en zoyl)p h en -
yl La u r a te (40). This benzophenone phosphate analogue was
prepared as described in procedure E (63% yield) and purified
by radial chromatography using 20% ethyl acetate in hexanes
to afford a white solid: mp 44-45 °C; Rf ) 0.55; 1H NMR (300
MHz, CDCl3) δ 7.83-7.79 (m, 4H), 7.31 (d, J ) 8.86 Hz, 2H),
7.21 (d, J ) 8.47 Hz, 2H), 2.59 (t, J ) 7.32 Hz, 2H), 1.77 (q, J
) 7.32 Hz, 2H), 1.53 (s, 18H), 1.27 (m, 16H), 0.88 (t, J ) 6.54
Hz, 3H).
1
a white solid: mp 65-67 °C; Rf ) 0.26; H NMR (300 MHz,
CDCl3) δ 7.44 (d, J ) 8.85 Hz, 2H), 7.32 (s, 10H), 6.99 (d, J )
8.09 Hz, 2H), 5.1 (d, J ) 8.47 Hz, 4H), 2.33 (t, J ) 7.7 Hz,
2H), 1.66 (q, J ) 7.7 Hz, 2H), 1.27 (s, 24H), 0.88 (t, J ) 6.55
Hz, 3H).
3-((Di(ben zyloxy)p h osp h or yl)oxy) P h en yl P a lm it a t e
(27). This resorcinol phosphate analogue was prepared as
described in procedure D (65% yield) and purified by radial
chromatography using 20% ethyl acetate in hexanes to afford
4-(4-((Di(ter t-b u t oxy)p h osp h or yl)oxy)b en zoyl)p h en -
yl Myr ista te (41). This benzophenone phosphate analogue
was prepared as described in procedure E (72% yield) and
purified by radial chromatography using 20% ethyl acetate in
1
a white solid: mp 45-46 °C; Rf ) 0.32; H NMR (300 MHz,
1
CDCl3) δ 7.32 (s, 10H), 7.30-6.91 (m, 4H), 5.12 (d, J ) 8.47
hexanes to afford a white solid: mp 54-55 °C; Rf ) 0.55; H

SARs of Lysophosphatidic Acid
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 6 969
1
NMR (300 MHz, CDCl3) δ 7.83-7.79 (m, 4H), 7.31 (d, J ) 8.86
Hz, 2H), 7.20 (d, J ) 8.47 Hz, 2H), 2.59 (t, J ) 7.7 Hz, 2H),
1.77 (q, J ) 7.32 Hz, 2H), 1.54 (s, 18H), 1.26 (m, 20H), 0.88 (t,
J ) 6.93 Hz, 3H).
2-(P a lm itoyla m in o)p h en yl Dih yd r ogen P h osp h a te (1).
This aminophenol phosphate analogue was deprotected as
described in procedure F (100% yield) affording a white solid:
mp 85-88 °C; 1H NMR (300 MHz, CDCl3/CD3OD (1:1)) δ 8.1-
6.95 (m, 4H), 2.28 (m, 2H), 1.62 (m, 2H), 1.22 (m, 24H), 0.82
(m, 3H).
mp 113-115 °C; H NMR (300 MHz, CDCl3) δ 7.77-7.19 (m,
4H), 2.56 (m, 2H), 1.69 (m, 2H), 1.24 (m, 20H), 0.84 (m, 3H).
Diben zyl (2,5,7,8-Tetr am eth yl-2-(4,8,12-tr im eth yldecyl)-
3,4-d ih yd r o-2H-6-ch r om en yl) P h osp h a te (42). This R-to-
copherol analogue was phosphorylated as described in proce-
dure D (84% yield) and purified by flash chromatography using
1
5% acetone in chloroform, affording a clear oil: Rf ) 0.48; H
NMR (300 MHz, CDCl3) δ 7.39 (m, 10H), 5.1 (m, 4H), 2.54 (t,
J ) 6.2 Hz, 2H), 2.18 (s, 3H), 2.15 (s, 3H), 2.06 (s, 3H), 1.76
(m, 1H), 1.57-1.07 (m, 22H), 0.88-0.83 (m, 15H).
2,5,7,8-Tetr a m eth yl-2-(4,8,12-tr im en tyld ecyl)-3,4-d ih y-
d r o-2H-6-cr om en yl Dih yd r ogen P h osp h a te (14). This
R-tocopherol phosphate analogue was deprotected as described
in procedure F (100% yield) affording a clear oil; 1H NMR (300
MHz, CDCl3) δ 2.54 (m, 2H), 2.18-2.09 (m, 9H), 1.76 (m, 1H),
1.57-1.07 (m, 22H), 0.88-0.83 (m, 15H).
3-(P a lm itoyla m in o)p h en yl Dih yd r ogen P h osp h a te (2).
This aminophenol phosphate analogue was deprotected as
described in procedure F (100% yield) affording a white solid:
mp 150 °C (dec); H NMR (300 MHz, CDCl3/CD3OD (1:1)) δ
7.36-6.85 (m, 4H), 2.24 (m, 2H), 1.6 (m, 2H), 1.22 (m, 24H),
0.82 (m, 3H).
1
Com pou n d P r epar ation . Phospholipids, which were stored
dissolved in organic solvent (usually 1:1 chloroform/methanol)
at -20° C under argon, were separated (0.1-2.0 µmol, 1-50
µL) into microcentrifuge tubes, dried in vacuo, and resus-
pended in water with 0.1% (w/v) fatty acid free BSA. After
brief vortexing, the mixture was treated for 5 min in a bath
sonicator. The solution was stored at room temperature for
no more than 2 h before use. Subsequent cycles of freezing/
thawing did not affect activity. In some cases, phospholipids
were suspended in buffer (HKRB, calcium measurements,
HEPES-buffered Dulbeco’s modified Eagle’s culture medium,
cAMP determinations).
4-(P a lm itoyla m in o)p h en yl Dih yd r ogen P h osp h a te (3).
This aminophenol phosphate analogue was deprotected as
described in procedure F (100% yield) affording a white solid:
mp 120 °C (dec); H NMR (300 MHz, CDCl3/CD3OD (1:1)) δ
7.5-7.05 (m, 4H), 2.28 (m, 2H), 1.62 (m, 2H), 1.25 (m, 24H),
0.82 (m, 3H).
1
3-(P h osp h on ooxy)p h en yl P a lm ita te (7). This resorcinol
phosphate analogue was deprotected as described in procedure
F (100% yield) affording a white solid: mp 79-82 °C; 1H NMR
(300 MHz, CDCl3/CD3OD (1:1)) δ 7.21-6.75 (m, 4H), 2.24 (m,
2H), 1.6 (m, 2H), 1.22 (m, 24H), 0.8 (m, 3H).
2-((Z)-9-Hecadecen oylam in o)ph en yl Dih ydr ogen P h os-
p h a te (4). This aminophenol phosphate analogue was depro-
tected as described in procedure G (100% yield) affording a
clear oil: 1H NMR (300 MHz, CDCl3/CD3OD (1:1)) δ 7.31-
6.98 (m, 4H), 5.36 (m, 2H), 2.31 (m, 2H), 1.94 (m, 4H), 1.62
(m, 2H), 1.26 (m, 18H), 0.88 (m, 3H).
3-((Z)-9-Hecadecen oylam in o)ph en yl Dih ydr ogen P h os-
p h a te (5). This aminophenol phosphate analogue was depro-
tected as described in procedure G (100% yield) affording a
white solid: mp 120 °C (dec); 1H NMR (300 MHz, CDCl3/
CD3OD (1:1)) δ 7.36-6.85 (m, 4H), 5.35 (m, 2H), 2.24 (m, 2H),
1.9 (m, 4H), 1.6 (m, 2H), 1.22 (m, 18H), 0.8 (m, 3H).
4-((Z)-9-Hecadecen oylam in o)ph en yl Dih ydr ogen P h os-
p h a te (6). This aminophenol phosphate analogue was depro-
tected as described in procedure G (100% yield) affording a
white solid: mp 140 °C (dec); 1H NMR (300 MHz, CDCl3/
CD3OD (1:1)) δ 7.5-7.05 (m, 4H), 5.35 (m, 2H), 2.3 (m, 2H),
1.9 (m, 4H), 1.65 (m, 2H), 1.22 (m, 18H), 0.82 (m, 3H).
3-(P h osph on ooxy)ph en yl (Z)-9-Hexadecen oate (8). This
resorcinol phosphate analogue was deprotected as described
in procedure G (100% yield) affording a clear oil: 1H NMR
(300 MHz, CDCl3/CD3OD (1:1)) δ 7.3-6.75 (m, 4H), 5.39 (m,
2H), 2.45 (m, 2H), 2.05 (m, 4H), 1.62 (m, 2H), 1.22 (m, 18H),
0.85 (m, 3H).
Cell Cu ltu r e. The LPA mimetics were tested on the human
breast cancer cell line MDA MB-231. The cells were grown in
Dubelco’s modified essential medium (DMEM) supplemented
in 10% FBS and were passaged following trypsinization every
5-7 days. Their growth medium was changed every third day.
Ca lciu m Assa y. Cell monolayers grown in 150 cm2 dishes
(>80% confluent) were overlaid with 10 mL of HKRB buffer
(20 mM HEPES, 103 mM NaCl, 4.8 mM KCl, 0.5 mM CaCl2,
1.2 mM MgSO4, 1.2 mM KH2PO4, 15 mM glucose, pH 7.4)
containing 5 µg/mL INDO-1AM. After 45 min at 37° C, the
monolayer was washed briefly with warm buffered saline, and
the cells were freed from the substratum by incubation with
trypsin/EDTA. After brief centrifugation, the cell pellet was
resuspended in HKRB at approximately 1.5 × 106 cells/mL
and kept at 37° C for no more than 90 min before use. Records
of free intracellular calcium were made on 2 mL aliquots of
cells suspended in a quartz cuvette in a temperature-controlled
fluorimeter (Aminco SLM 800; SLM Instruments, Urbana, IL).
Excitation was with light at 332 nm (slit width 4 nm), and
emission wavelengths were 400 and 485 nm (slit widths 4 nm).
Calcium responses recorded were peak responses in traces of
the ratio of the emitted light. Maximum and minimum
fluorescence ratios were calculated by the sequential addition
of digitonin (to 75 µM) and EDTA (to 5 µM). Calcium mobiliza-
tion was presented as a percent of the total available calcium
(defined by subsequent treatment with digitonin) normalized
to maximal LPA response, which is typically 30% of the total
amount of calcium mobilized by digitonin. All points used to
calculate the EC50 values were done in triplicate and were
curved-fitted with GraphPAD (GraphPAD software, San Diego,
CA).
4-(4-(P h osp h on ooxy)ben zoyl)p h en yl Hexa n oa te (9).
This benzophenone phosphate analogue was deprotected as
described in procedure H (100% yield) affording a white solid:
1
mp 99-100 °C; H NMR (300 MHz, CDCl3) δ 7.81-6.95 (m,
4H), 2.57 (m, 2H), 1.83 (m, 2H), 1.38 (m, 4H), 0.89 (m, 3H).
4-(4-(P h osp h on ooxy)ben zoyl)p h en yl Octa n oa te (10).
This benzophenone phosphate analogue was deprotected as
described in procedure H (100% yield) affording a white solid:
1
mp 101-103 °C; H NMR (300 MHz, CDCl3) δ 7.72-7.12 (m,
Ack n ow led gm en t. The authors thank Doug Be-
shore and Sonya Carter for the summer they spent
doing preliminary studies and Ben Brooks for his help
in obtaining elemental analysis. This work was sup-
ported by a grant from the National Institute of General
Medical Sciences (RO1-GM52722).
4H), 2.55 (m, 2H), 1.75 (m, 2H), 1.34 (m, 8H), 0.89 (m, 3H).
4-(4-(P h osp h on ooxy)ben zoyl)p h en yl Deca n oa te (11).
This benzophenone phosphate analogue was deprotected as
described in procedure H (100% yield) affording a white solid:
1
mp 105-106 °C; H NMR (300 MHz, CDCl3) δ 7.76-7.15 (m,
4H), 2.55 (m, 2H), 1.71 (m, 2H), 1.27 (m, 12H), 0.88 (m, 3H).
4-(4-(P h osp h on ooxy)ben zoyl)p h en yl La u r a te (12). This
benzophenone phosphate analogue was deprotected as de-
scribed in procedure H (100% yield) affording a white solid:
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4-(4-(P h osp h on ooxy)ben zoyl)p h en yl Myr ista te (13).
This benzophenone phosphate analogue was deprotected as
described in procedure H (100% yield) affording a white solid:

970 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 6
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