Synthesis and pharmacological evaluation of second-generation phosphatidic acid derivatives as lysophosphatidic acid receptor ligands

Article abstract of DOI:10.1016/j.bmcl.2005.10.031

Short-chain phosphatidic acid derivatives, dioctanoyl glycerol pyrophosphate (DGPP 8:0, 1) and phosphatidic acid 8:0 (PA 8:0, 2), were previously identified as subtype-selective LPA₁ and LPA₃ receptor antagonists. Recently, we report

Full text of DOI:10.1016/j.bmcl.2005.10.031

Bioorganic & Medicinal Chemistry Letters 16 (2006) 633–640
Synthesis and pharmacological evaluation of
second-generation phosphatidic acid derivatives as
lysophosphatidic acid receptor ligands
Gangadhar G. Durgam,a Ryoko Tsukahara,b Natalia Makarova,b Michelle D. Walker,b
Yuko Fujiwara,b Kathryn R. Pigg,c Daniel L. Baker,c Vineet M. Sardar,d Abby L. Parrill,d
Gabor Tigyib and Duane D. Millera,*
aDepartment of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee
Health Science Center, Memphis, TN 38163, USA
bDepartment of Physiology, College of Medicine, University of Tennessee Health Science Center, Memphis, TN 38163, USA
cDepartment of Medicine, College of Medicine, University of Tennessee Health Science Center, Memphis, TN 38163, USA
dDepartment of Chemistry, Computational Research Institute on Materials, University of Memphis, Memphis, TN, USA
Received 19 September 2005; revised 8 October 2005; accepted 12 October 2005
Available online 2 November 2005
Abstract—Short-chain phosphatidic acid derivatives, dioctanoyl glycerol pyrophosphate (DGPP 8:0, 1) and phosphatidic acid 8:0
(PA 8:0, 2), were previously identified as subtype-selective LPA1 and LPA3 receptor antagonists. Recently, we reported that the
replacement of the phosphate headgroup by thiophosphate in a series of fatty alcohol phosphates (FAP) improves agonist as well
as antagonist activities at LPA GPCR. Here, we report the synthesis of stereoisomers of PA 8:0 analogs and their biological eval-
uation at LPA GPCR, PPARc, and ATX. The results indicate that LPA receptors stereoselectively interact with glycerol backbone
modified ligands. We observed entirely stereospecific responses by dioctyl PA 8:0 compounds, in which (R)-isomers were found to be
agonists and (S)-isomers were antagonists of LPA GPCR. From this series, we identified compound 13b as the most potent LPA3
receptor subtype-selective agonist (EC50 = 3 nM), and 8b as a potent and selective LPA3 receptor antagonist (Ki = 5 nM) and inhib-
itor of ATX (IC50 = 600 nM). Serinediamide phosphate 19b was identified as an LPA3 receptor specific antagonist with no effect on
LPA1, LPA2, and PPARc.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Lysophosphatidic acid (LPA, 1-or 2-acyl-sn-glycero-3-
phosphate) is a pleiotropic growth factor-like lipid medi-
ator with diverse biological properties. LPA elicits
numerous cellular responses including cell proliferation,
cell survival, cell migration, platelet aggregation,
smooth muscle contraction, cytokine and chemokine
secretion, and neurite retraction in various cell types.1,2
LPA mediates its effects through the activation of three
G protein-coupled receptors (GPCR), LPA1, LPA2, and
LPA3, which belong to endothelial differentiation gene
family.3 LPA4, formerly known as the P2Y9/GPR23
orphan GPCR, has recently been identified as a fourth
plasma membrane LPA receptor.4 LPA4 is evolutionari-
ly distant and shares only 20–24% homology with LPA1,
LPA2, and LPA3.4 Recently, the nuclear transcription
factor peroxisome proliferator-activator receptor-c
(PPARc) has been identified as an intracellular receptor
for LPA.5
LPA signaling pathways are involved in several physio-
logical and pathological processes including cancer inva-
sion,2,6 atherogenesis,7,8 inflammation,9 angiogenesis,10
neurogenesis,11 wound healing,12,13 protection against
radiation- and chemotherapy-induced apoptosis,14,15
and ischemia–reperfusion injury.16 The autocrine motil-
ity factor autotaxin (ATX), originally isolated from
invasive cancer cells, has recently been identified as the
long elusive plasma lysophospholipase D activity17,18
that is the major source of LPA production in serum19,20
and tumor cells.18 ATX expression increases motility
and invasiveness of tumor cells in vitro via the produc-
tion of LPA and subsequent activation of LPA1.21 Thus,
Keywords: LPA; Phosphatidic acid; Autotaxin.
*
Corresponding author. Tel.:+ 1 901 448 6026; fax:+1 901 448
3446; e-mail: dmiller@utmem.edudmiller@utmem.edu
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.10.031

634
G. G. Durgam et al. / Bioorg. Med. Chem. Lett. 16 (2006) 633–640
LPA receptor agonists, antagonists, and compounds
that block LPA production via ATX are likely to have
great therapeutic potential. Despite the potential thera-
peutic applications of LPA receptor agonists and antag-
onists, a limited number of subtype-selective agents have
been reported to date.3,22 Furthermore, the agents that
have been described in the literature have not been com-
prehensively characterized at each LPA target.
stereoselective preference for (R)-1-alkyl glycerophos-
phate 18:1 (AGP) over (S)-3AGP 18:1.8 Thus, streo-
chemically pure analogs are preferred over racemic
mixtures as the enantiomers are likely to possess differ-
ent pharmacological properties.
Based on the observation that shorter-chain LPA ana-
logs exert little or no activity on LPA receptors,24,32
we have identified previously short-chain phosphatidic
acid derivatives, dioctanoyl glycerol pyrophosphate
(DGPP 8:0, 1) and phosphatidic acid 8:0 (PA 8:0, 2),
as subtype-selective LPA1 and LPA3 receptor antago-
nists (Fig. 1).33 Recently, we reported that the replace-
ment of the phosphate headgroup by thiophosphate in
a series of fatty alcohol phosphates (FAP) improves
agonist as well as antagonist activities at LPA GPCR.34
Here, we report the synthesis of stereoisomers of PA 8:0
analogs and their biological evaluation at LPA GPCR,
PPARc, and ATX. Our present data extend previous re-
ports,28,30,31 in that LPA receptors stereoselectively
interact with glycerol backbone modified ligands. With
dioctyl PA 8:0 compounds, we observed entirely stereo-
specific responses, in which (R)-isomers were found to
be agonists, whereas (S)-isomers were antagonists of
LPA GPCR. From this series, we identified compound
13b as the most potent LPA3 receptor subtype-selective
agonist (EC50 = 3 nM), and 8b as a potent and selective
LPA3 receptor antagonist (Ki = 5 nM) and inhibitor of
ATX (IC50 = 600 nM). Serinediamide phosphate 19b
was identified as an LPA3 receptor specific antagonist
with no effect on LPA1, LPA2, and PPARc. These com-
pounds therefore offer much improved selectivity and
potency over the first generation of compounds for
manipulating the biological responses mediated through
the different targets of LPA.
Several groups have reported that LPA receptors, unlike
many other GPCR, lack stereospecificity or stereoselec-
tivity in recognition of acyl- and alkyl-LPA analogs.23–27
In contrast to the natural ligand LPA, LPA receptors
have been shown to interact stereoselectively with
backbone modified LPA analogs, in which the glycerol
backbone has been replaced by serine or ethanolamine.
Heise et al. showed that LPA receptors prefer the natu-
ral (R)-stereochemistry in a 2-substituted ethanolamide
series of compounds.28 Although both enantiomers were
equally effective inhibitors of LPA-activated chloride
currents in Xenopus laevis oocytes, (S)-N-palmitoyl ser-
ine phosphoric acid was more potent than the (R)-enan-
tiomer in eliciting calcium mobilization in MDA MB231
cells.29,30 Recently, (2S)-1-oleoyl-2-O-methyl-glycero-
phosphothionate (OMPT) was found to be more potent
than (2R)-OMPT in calcium mobilization assays using
transfected rat hepatoma RH7777 and insect Sf9 cells.31
In contrast to the plasma membrane receptors, activa-
tion of the intracellular LPA receptor PPARc shows a
O
O
RO P O
OH
O
O
O
R
Dioctanoyl PA analogs were synthesized as shown in
Scheme 1. Commercially available (2S)-3-benzyloxy-
1,2-propanediol (3a) was diacylated with octanoyl-
chloride followed by debenzylation under catalytic
(1) DGPP 8:0 P(O)(OH)2
(2) PA 8:0
H
Figure 1. Structures of DGPP 8:0 (1) and PA 8:0 (2).
O
O
C7H15
OH
b or c
a
HO
O
C7H15
BnO
OH
4a
(2S)-3a
O
O
O
X
RO P O
OR
O
C7H15
X
O
C7H15
d or e
O
C7H15
HO P O
OH
O
C7H15
O
O
X
R
X
7a
8a
O
S
5a
6a
O
S
Bn
CH2CH2CN
O
OH
S
HO P O
OH
O
C7H15
O C7H15
4 steps
BnO
OH
(2R)-3b
8b
O
Scheme 1. Synthesis of dioctanoyl PA analogs. Reagents and conditions: (a) (i) C7H15COCl, pyridine, CH2Cl2, 70%; (ii) Pd–C/H2, MeOH, 95%; (b)
(i) dibenzyl-N,N-diisopropyl phosphoramidite, 1H-tetrazole, CH2Cl2; (ii) 50% H2O2, 60%; (c) (i) bis(2-cyanoethyl)-N,N-diisopropyl phosphorami-
dite, 1H-tetrazole, CH2Cl2; (ii) S, reflux, 72%; (d) Pd–C/H2, MeOH, 80%; (e) bis(trimethylsilyl)trifluoro acetamide, DBU, pyridine, 40%.

G. G. Durgam et al. / Bioorg. Med. Chem. Lett. 16 (2006) 633–640
635
hydrogenation conditions to provide the alcohol (4a).
Alcohol (4a) was then phosphorylated using dibenzyl-
N,N-diisopropyl phosphoramidite to yield the dibenzyl
protected phosphate after oxidation (5a),35 which upon
catalytic hydrogenation afforded the corresponding
(2S)-dioctanoyl PA (7a). Treatment of 4a with bis(2-
cyanoethyl)-N,N-diisopropyl phosphoramidite followed
by reflux in presence of elemental sulfur provided the dicy-
anoethyl protected thiophosphate (6a). The target thio-
phosphatidic acid 8:0 (TPA 8:0, 8a) was obtained by
removing the cyanoethyl groups using bis(trimethylsi-
lyl)trifluoro acetamide and pyridine.36 Similarly, the
(2R)-thiophosphate analog 8b was synthesized from
(2R)-3-benzyloxy-1,2-propanediol (3b) in 4 steps. The
(2R)-dioctanoyl PA compound (2) used in this study
was purchased from commercial sources.
The dialkyl PA 8:0 (APA 8:0) analogs were synthesized
as shown in Scheme 2. Alkylation of commercially avail-
able (2S)-3-benzyloxy-1,2-propanediol (3a) with octylbr-
omide followed by debenzylation provided the alcohol
(9a). Compound 9a was then phosphorylated using
phosphoramidite chemistry to produce the di-tert-butyl
protected phosphate (10a),35 which upon treatment with
TFA gave the corresponding (2S)-dioctyl PA com-
pound (12a). Treatment of 9a with bis(2-cyanoethyl)-N,
N-diisopropyl phosphoramidite followed by reflux in
presence of elemental sulfur provided the dicyanoethyl
protected thiophosphate (11a). Removal of the cyano-
ethyl groups with treatment of KOH in methanol fur-
nished the target dialkyl thiophosphatidic acid 8:0
(ATPA 8:0) compound 13a. Similarly, the (2R)-analogs
12b and 13b were synthesized from (2R)-3-benzyloxy-
1,2-propanediol (3b) in 4 steps.
OC8H17
OH
b or c
a
HO
OC8H17
BnO
OH
9a
(2S)-3a
X
HO P
OH
OC8H17
X
OC8H17
OC8H17
d or e
O
OC8H17
RO P
O
OR
X
O
S
R
Bn
X
O
S
12a
13a
10a
11a
CH2CH2CN
The serinediamide phosphate/thiophosphate (SDP/
SDTP) analogs were synthesized as outlined in Scheme
3. O-Benzyl-Boc-(L)-serine (14a) was coupled with octyl-
amine using EDC and HOBt, and deprotection with
TFA gave compound 15a. Compound 15a was acylated
using octanoyl chloride followed by debenzylation to
yield the key alcohol intermediate (16a). The alcohol
(16a) was then phosphorylated to yield the target (2S)-
compounds 19a and 20a via formation of intermediates
17a and 18a, using chemistry similar to that shown in
Scheme 1. From O-benzyl-Boc-(D)-serine (14b) as the
starting material, the (2R)-analogs 19b and 20b were
OH
X
OC8H17
4 steps
BnO
OH
HO P
O
OC8H17
OH
X
(2R)-3b
12b O
13b S
Scheme 2. Synthesis of dioctyl PA analogs. Reagents and conditions:
(a) (i) C8H17Br, KOH, toluene, reflux, Dean–Stark apparatus, 65%; (ii)
Pd–C/H2, MeOH 92%; (b) (i) di-tert-butyl-N,N-diisopropyl phospho-
ramidite, 1H-tetrazole, CH2Cl2; (ii) 50% H2O2, 68%; (c) (i) bis(2-
cyanoethyl)-N,N-diisopropyl phosphoramidite, 1H-tetrazole, CH2Cl2;
(ii) S, reflux, 73%; (d) TFA, CH2Cl2, 84%; (e) KOH/MeOH, rt, 3h,
46%.
O
O
O
a
b
HO
NHC8H17
C7H15
16a
BnO
OH
NHBoc
BnO
O
NHC8H17
HN
O
NH2.CF3COOH
O
(2S)-14a
15a
X
X
RO P O
OR
HO P O
OH
NHC8H17
C7H15
c or d
e or f
NHC8H17
C7H15
HN
HN
O
O
X
O
S
R
Bn
X
O
S
19a
20a
17a
18a
CH2CH2CN
O
O
O
X
6 steps
HO P O
OH
NHC8H17
C7H15
BnO
OH
NHBoc
HN
X
O
S
(2R)-14b
19b
20b
Scheme 3. Synthesis of serinediamide phosphate and thiophosphate analogs. Reagents: (a) (i) C8H17NH2, EDC/HOBt, CH2Cl2, 92%; (ii) TFA,
CH2Cl2, 90%; (b) (i) octanoyl chloride, NMM, THF, 82%; (ii) Pd–C/H2, MeOH, 90%; (c) (i) dibenzyl-N,N-diisopropyl phosphoramidite, 1H-
tetrazole, CH2Cl2; (ii) 50% H2O2, 68%; (d) (i) bis(2-cyanoethyl)-N,N-diisopropyl phosphoramidite, 1H-tetrazole, CH2Cl2; (ii) S, reflux, 66%; (e) Pd–
C/H2, MeOH, 80%; (f) bis(trimethylsilyl)trifluoro acetamide, DBU, pyridine, 38%.

636
G. G. Durgam et al. / Bioorg. Med. Chem. Lett. 16 (2006) 633–640
1
synthesized. All compounds were characterized by H
NMR, mass spectroscopy and, in case of the final com-
pounds, elemental analysis.37
manner. To address this hypothesis, we synthesized
and evaluated several PA 8:0 analogs. The effects of
headgroup modification to a thiophosphate, glycerol
backbone modification to serine, as well as the impor-
tance of the ester versus ether linkage of hydrophobic
chain and stereochemistry are assessed in the current
study using a constant octyl hydrophobic chain length
that was previously found to be optimal.
Thiophosphates 8a–b, 13a–b, and 20a–b were built with
phosphate groups in the À2 ionization state in the
MOE38 molecular modeling software package. Partial
charges were assigned and structures were geometry
optimized using the MMFF9439 forcefield. Docking
studies with active and inactive models of the LPA
receptors were performed using the Autodock 3.0 soft-
ware40 as previously described for DGPP 8:0.41 Default
parameters were used with the exception of the number
of energy evaluations (9 · 1010), generations (30,000),
local search iterations (3000), and runs (15). Docking
boxes included the extracellular loops and top of the
transmembrane domains of the inactive receptor models
and the transmembrane domains of the active receptor
models. Complexes described in the Results section are
those for each receptor:ligand pair with the lowest final
docked energy. Residues are described using the helix
indexing system of Ballesteros and Weinstein.42 This
system assigns numbers in the format of X.Y to residues
in the transmembrane domains of GPCR using the
transmembrane helix number (X) and an index relative
to the most conserved residue in that helix assigned
index position 50 (Y).
All synthesized analogs were tested for agonist and
antagonist activities at LPA GPCR, as activators of
PPARc and as inhibitors of ATX. RH7777 cells, which
are devoid of LPA GPCR, were stably transfected with
individual LPA1, LPA2, and LPA3 receptors and used
for in vitro screening.33,44 PPARc activation was as-
sayed in CV1 cells transfected with PPARc and an
acyl-coenzyme A oxidase-luciferase (PPRE-Acox-Rluc)
reporter gene.8 ATX inhibition was determined using
bis(para-nitrophenyl) phosphate hydrolysis by recombi-
nant ATX in conditioned medium from transiently
transfected HEK293 cells as described previously.45
These results are shown in Table 1.
(2S)-PA 8:0 (7a) and its enantiomer (2R)-PA 8:0 (2)
showed subtype selective antagonism at the LPA1 and
LPA3 receptors with no effect on the LPA2 receptor.
PA analogs enantioselectively antagonized both LPA1
and LPA3 receptors with a moderate preference for the
S-isomer at LPA1. The antagonist selectivity is reversed
at the LPA3 receptor, which showed a preference for
(2R)-PA 8:0 over the (2S)-isomer. In accordance with
previous reports,46 thiophosphate headgroup modifica-
tion improved the activity of PA 8:0 analogs. (2R)-TPA
8:0 (8b) was a more potent antagonist than the phosphate
analog (2R)-PA 8:0 (2) at both the LPA1 and LPA3
receptors, and was a partial agonist of LPA2. Compound
8b was identified as the most potent and selective LPA3
receptor antagonist reported to date with a Ki value of
5 nM with a 75-fold selectivity over LPA1.
We reported earlier DGPP 8:0 (1) and PA 8:0 (2) as sub-
type selective antagonists of the LPA1 and LPA3 recep-
tors with an order of magnitude preference for LPA3.33
However, these compounds were derived from the natu-
ral sources and were available only in (R)-enantiomeric
form. The activities of the (S)-enantiomers have not pre-
viously been assessed at LPA GPCR. Since the discov-
ery of PA 8:0 as an LPA1/3 antagonist, there have
been many advances in the LPA field. LPA receptors,
which were previously reported to have no stereochem-
ical preference for the natural ligand LPA,23,24 have now
been shown to interact stereoselectively with synthetic
ligands, in which the glycerol backbone is either
modified or has a substitution at the sn-2 posi-
tion.28,30,31,43 We hypothesized that the PA 8:0 scaffold
would interact with LPA receptors in a stereoselective
The modeled complex of 8b with the inactive model of
the previously validated LPA3 receptor47 is shown in
Figure 2A. This model shows significantly stronger
interactions between 8b and the receptor than did the
Table 1. Effects of PA 8:0 analogs on LPA1–3 transfected RH7777 cells, activation of PPARc, and ATX inhibition
Compound
R/S
X
LPA1
LPA2
LPA3
ATX
a
EC50 (Emax
(nM)
)
IC50 (Ki)
(nM)
EC50 (Emax
(nM)
)
IC50 (Ki)
(nM)
EC50 (Emax
(nM)
)
IC50 (Ki)
(nM)
IC50 (nM)
(Max. inhib. %)
7a
2
S
R
S
R
S
R
S
R
S
R
S
R
O
O
S
NEb
NE
433 (221)
692 (407)
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
207 (119)
85 (39)
NE
NE
1644 (49.9)
727 (52.3)
597 (72.5)
NE
8a
8b
NE
NE
7170 (17)
6330 (58)
NE
115 (30)
NE
NE
S
686 (360)
1580 (486)
NE
11 (5)
143 (50)
NE
12a
12b
13a
13b
19a
19b
20a
20b
O
O
S
NE
3260 (57)
NE
NE
NE
164 (109)
NE
NE
NE
328 (139)
NE
NE
184 (67)
NE
S
695 (30)
NE
NE
5720 (27)
NE
NE
3 (109)
NE
NE
NE
NE
O
O
S
414 (196)
935 (489)
251 (117)
302 (118)
NE
476 (152)
7390 (2850)
NE
NE
NE
NE
NE
NE
NE
NE
S
NE
a Emax = maximal efficacy of the drug/maximal efficacy of LPA 18:1, expressed as the percentage.
b NE = no effect.

G. G. Durgam et al. / Bioorg. Med. Chem. Lett. 16 (2006) 633–640
637
(12a–b and 13a–b) and evaluated their agonist and antag-
onist properties at LPA GPCR. None of the compounds
evoked Ca2+ transients in wild-type RH7777 cells. In
general, thiophosphates were more potent than the cor-
responding phosphates regardless of agonist/antagonist
activity. We observed enantiospecific activation of the
LPA1/3 receptors by (2R)-APA 8:0 (12b) and (2R)-ATPA
8:0 (13b). Compound 13b, which has the identical (R)-
configuration as natural LPA, was the most potent and
LPA3-selective agonist in this series (EC50 = 3 nM,
Emax = 109%) and was ꢀ230- and ꢀ1900-fold selective
for LPA3 over LPA1 and LPA2, respectively. The high
potency of 13b is evident from the molecular model
shown in Figure 2B. The thiophosphate headgroup
forms ion pairs with R(105)3.28, R(236)7.36, K(235)
7.35, and R(185)5.38. The ion pair with R(236)7.36 is
unique to this thiophosphate due to the greater phospho-
rus–sulfur bond length compared to that of phosphorus–
oxygen. In fact, mutagenesis of LPA3 demonstrates that
the R(236)7.36A mutant maintains activation by LPA,
demonstrating that the phosphate of LPA does not form
the same ion pair observed for the thiophosphate.47 The
additional ion pair explains the enhanced potency of this
and other thiophosphate agonists of LPA3. At the LPA3
receptor, dioctyl thiophosphate analog 13b was a more
potent agonist than the corresponding phosphate (12b)
and LPA 18:1 (Fig. 3). In contrast to the (2R)-alkyl ana-
logs, the (2S)-enantiomers were antagonists at the LPA1/
receptors. Although compounds (R)-VPC12204 and
3
(S)-VPC12249 were the first to demonstrate enantiospec-
ific agonist and antagonist responses, respectively, at
LPA1, both enantiomers were antagonists at LPA3.28
The enantiosepecific activation of the LPA1/3 receptors
by APA analogs is presumably due to the favorable
orientation of the conformationally flexible alkyl side
chains of the (R)-isomers within the ligand binding
pocket of the receptors. The side chains of acyl PA ana-
logs are relatively constrained due to their ester linkage
to glycerol, which may prevent these favorable ligand–re-
ceptor interactions. With the exception of 2(R)-ATPA
8:0 (13b), which was a weak partial agonist of LPA2,
all alkyl PA analogs tested had no effect on LPA2
receptor.
Figure 2. Computational models of LPA3 complexes. Ribbons repre-
sent portions of the LPA3 receptor colored from red at the amino
terminal end to blue at the carboxy terminal end. Synthetic ligands are
shown as ball and stick models. Key residues of LPA3 interacting with
synthetic ligands are shown as stick models and labeled. (A) Best
docked position of 8b in the inactive model of LPA3. (B) Best docked
position of 13b in the active model of LPA3.
complex obtained using the active model of the LPA3
receptor. Figure 2A shows ionic interactions between
the thiophosphate group of 8b and R(105)3.28,
R(276)7.36, and K95. A hydrogen bond involving
Q(106)3.29 and 8b is also observed. The ionic interac-
tions are consistent with our previously reported model
of DGPP 8:0 with the inactive model of LPA3.41
The hydrogen bonding interaction with Q(106)3.29,
however, is unique to compound 8b and may explain
its exceptional potency. The compound (2S)-TPA 8:0
(8a) was devoid of LPA1/3 antagonism, but was a partial
agonist at LPA2 and LPA3. The models of 8a with both
the inactive and active models of LPA3 (data not shown)
showed relatively poor ionic interactions with the
receptor, indicating that the stereochemical difference
prevents the thiophosphate group from interacting with
the key residues required for either full agonist or
antagonist activity. These results, in accordance with
the previously published reports,31 show that LPA
receptors exhibit stereoselectivity in interacting with
sn-2 substituted glycerol analogs.
Replacement of the glycerol backbone by serine is well
tolerated at LPA GPCR.29,30,48 Surprisingly, SDP 8:0
analogs (19a–b) were identified as LPA3 receptor sub-
type-specific antagonists that had no effect on LPA1 and
LPA2. As expected, SDP 8:0 isomers also demonstrated
enantioselective LPA3 antagonism. The (S)-isomer (19a)
was a better antagonist than was the (R)-isomer (19b).
Thiophosphate headgroup modification in serinedia-
mides (20a–b) not only improved the LPA3 antagonist
activity but also resulted in loss of LPA3 subtype-specific-
ity by conferring added LPA1 antagonism.
Results of in vitro PPARc activation by these com-
pounds are shown in Figure 4. Zhang et al. reported that
PPARc showed a preference for alkyl-LPA over acyl-
LPA and the R-isomer over the S-isomer in the AGP
18:1 enantiomeric pair.8 In our series, only alkyl-PA
analogs elicited PPARc activation, while acyl-PA and
serinediamide analogs had no activity (Fig. 4). Unlike
To increase the stability of the acyl-PA 8:0 analogs
against chemical as well as phospholipase A (PLA) deg-
radation, we synthesized alkyl derivatives of PA 8:0

638
G. G. Durgam et al. / Bioorg. Med. Chem. Lett. 16 (2006) 633–640
Acox-PPRE-Rluc fold induction + s.e.
A
LPA1
0
1
2
3
4
5
6
120
100
80
60
40
20
0
LPA 18:1
13b
12b
vehicle
Rosiglitazone
*
LPA 20:4
7a
*
2
8a
8b
12a
12b
13a
13b
19a
19b
20a
20b
*
*
*
*
*
Figure 4. In vitro PPARc activation by PA analogs in CV1 cells
transfected with PPARc and PPRE-Acox-Rluc reporter genes. Effects
are compared with rosiglitazone, a known PPARc agonist. CV1 cells
were treated with vehicle or 10 lM test compound dissolved in DMSO
for 20 h. Luciferase and b-galactosidase activities (mean SEM) were
measured in the cell lysate (n = 4). *P < 0.05, significant differences
over vehicle control.
10
100
1000
10 4
10 5
Concentration (nM)
B
LPA3
120
100
LPA 18:1
13b
12b
inhibition. Likewise, dialkyl substitution (13a–b) in the
thiophosphate series has resulted in loss of ATX inhibito-
ry activity. All four compounds containing the serine
backbone showed no inhibition of ATX.
80
60
40
20
0
In summary, a series of PA analogs has been synthesized
and evaluated at LPA GPCR, PPARc, and ATX. Our
results substantiate previously published reports that
LPA receptors show marked enantioselectivity with
phospholipid analogs having sn-2 substituted glycerol
or serine backbones. Enantioselectivity is determined,
not only by the backbone but also by the nature of
the hydrocarbon chain linkage and stereochemistry of
the ligands. These results indicate that fine-tuning the
structural features of the available lead structures will
aid in development of LPA receptor type- and sub-
type-specific ligands. This study has resulted in identifi-
cation of compound 13b as a potent LPA3 receptor
subtype-selective agonist and compound 8b (Ki = 5 nM)
as the most potent subtype-selective LPA3 receptor
antagonist reported to date. In addition, compound 8b
blocks LPA1 and ATX, two proteins linked to cancer
cell invasion and metastasis. Finally, using serine as a
glycerol backbone substitute, an LPA3 receptor-specific
antagonist 19b was discovered that has no effect on
LPA1, LPA2, and PPARc.
0.1
1
10
100
1000
104
10 5
Concentration (nM)
Figure 3. (2R) Alkyl PA analogs (12b and 13b) are agonists at LPA1
and LPA3 receptors expressed in RH7777 cells. Dose–response
relationships for LPA 18:1, 12b and 13b in RH7777 cells expressing
LPA1 (A) and LPA3 (B). Intracellular Ca2+ transients were measured
in response to the application of increasing concentrations of
compounds 12b and 13b, and compared to transients elicited by
LPA 18:1. Data points represent the average of four measurements.
the enantiospecific responses at LPA GPCR, there was
no stereoselectivity observed in PPARc activation by
these analogs. Compound 19a, which is a selective
LPA3 antagonist with no effect on LPA1/2, also showed
PPARc activation. In contrast, 19b retains LPA3 recep-
tor selectivity and had no effect on PPARc making it a
true LPA3 receptor specific antagonist.
Acknowledgment
This research was supported by a grant from the
National Institutes of Health (CA 92160).
ATX inhibition was noted for only acyl thiophosphates
8a and 8b (Table 1). The (R)-isomer (8b) was slightly more
potent and effective than its corresponding (S)-isomer
(8a). Modification of the thiophosphate headgroup
by phosphate (2 and 7a) significantly decreased ATX
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