Synthesis and pharmacological evaluation of the stereoisomers of 3-carba cyclic-phosphatidic acid

Article abstract of DOI:10.1016/j.bmcl.2010.09.115

Cyclic phosphatidic acid (CPA) is a naturally occurring analog of lysophosphatidic acid (LPA) in which the sn-2 hydroxy group forms a five-membered ring with the sn-3 phosphate. Here, we describe the synthesis of R-3-CCPA and S-3-CCPA along with their pha

Full text of DOI:10.1016/j.bmcl.2010.09.115

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7525–7528
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and pharmacological evaluation of the stereoisomers
of 3-carba cyclic-phosphatidic acid
Renuka Gupte a, Anjaih Siddam a, Yan Lu a, Wei Li a, Yuko Fujiwara b, Nattapon Panupinthu d,
Truc-Chi Pham c, Daniel L. Baker c, Abby L. Parrill c, Mari Gotoh e, Kimiko Murakami-Murofushi e,
Susumu Kobayashi f, Gordon B. Mills d, Gabor Tigyi b, Duane D. Miller a,
⇑
a Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, Memphis, TN 38163, USA
b Department of Physiology, College of Medicine, University of Tennessee Health Science Center, Memphis, TN 38163, USA
c Department of Chemistry, University of Memphis, TN 38152, USA
d Department of Systems Biology, M. D. Anderson Cancer Center, The University of Texas, Houston, TX 77054, USA
e Department of Biology, Ochanomizu University, Tokyo 112-8610, Japan
f Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba 278-8510, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 June 2010
Revised 22 September 2010
Accepted 23 September 2010
Available online 8 October 2010
Cyclic phosphatidic acid (CPA) is a naturally occurring analog of lysophosphatidic acid (LPA) in which the
sn-2 hydroxy group forms a five-membered ring with the sn-3 phosphate. Here, we describe the synthe-
sis of R-3-CCPA and S-3-CCPA along with their pharmacological properties as inhibitors of lysophospho-
lipase D/autotaxin, agonists of the LPA5 GPCR, and blockers of lung metastasis of B16-F10 melanoma cells
in a C57BL/6 mouse model. S-3CCPA was significantly more efficacious in the activation of LPA5 com-
pared to the R-stereoisomer. In contrast, no stereoselective differences were found between the two iso-
mers toward the inhibition of autotaxin or lung metastasis of B16-F10 melanoma cells in vivo. These
results extend the potential utility of these compounds as potential lead compounds warranting evalua-
tion as cancer therapeutics.
Keywords:
Lysophosphatidic acid
NPP2
Autotaxin
GPR92
Ó 2010 Elsevier Ltd. All rights reserved.
Lysophospholipase D
Lysophosphatidic acid (LPA) is a pleiotrophic phospholipid
growth factor with multiple roles in cancer metastasis and
progression.1 LPA elicits numerous biological effects including
the promotion of cellular survival, mitogenesis, angiogenesis,
migration, and cancer invasion that are mediated, at least in part,
by specific cell surface G-protein coupled receptors (GPCR) and
intracellular targets that include the nuclear hormone receptor
LPA2 GPCR.5 Substitution of the sn-2 or sn-3 oxygen with a methy-
lene in CPA yields carba-CPA (CCPA), a stabilized analog of CPA.6
Previous work has shown that 3-CCPA does not activate the
LPA1-4 GPCR5 but is a weak agonist of LPA5.7
Autotaxin (ATX) was initially identified as an autocrine tumor
cell motility factor from melanoma cell conditioned medium.8 ATX
has lysophospholipase D enzyme activity and is responsible for the
hydrolysis of lysophophatidylcholine leading to the generation of
LPA9,10 and CPA.11 While ATX can also produce sphingosine-1-phos-
phate (S1P) in vitro, it does not appear to contribute in a major way
to S1P production in vivo. High levels of autotaxin are generated by a
wide variety of metastatic human tumor cell lines including human
teratocarcinoma,12 hepatocellularcarcinoma,13 metastatic breast
cancer,1 ovariancancer,14 thyroid carcinoma,15 prostate cancer,16
follicular lymphoma17 and glioblastomamultiforme.18 ATX also
plays an important role in the chemotherapeutic resistance of
breast19 and ovarian cancer cells14 to chemotherapeutic agents.
ATX is under feedback inhibition by its hydrolytic products LPA,
CPA, and sphingosine-1-phosphate (S1P).20 Racemic 2-CCPA and
3-CCPA are potent inhibitors of ATX activity and 3-CCPA has been
shown to reduce lung metastasis of B16-F10 melanoma cells in-
jectedintravenously into C57BL/6mice.5 To further explorethe ther-
apeutic utility of 3-CCPA, stereochemically pure isomers are needed.
peroxisome proliferator-activated receptor (PPARc
).2 Cylic-phos-
phatidic acid (1-acyl-2,3-glycerophosphate, CPA) is a naturally
occurring analog of LPA in which the sn-2 hydroxy group forms a
five-membered ring with the sn-3 phosphate.3 CPA affects numer-
ous cellular functions, including inhibition of cell cycle progres-
sion, induction of stress fiber formation, inhibition of tumor cell
invasion and metastasis, and regulation of differentiation and
survival of neuronal cells.4 CPA is a weak agonist of the LPA1 and
Abbreviations: ATX, autotaxin; BSA, bovine serum albumin; CCPA, carbacyclic
phosphatidic acid; CPA, cyclic phosphatidic acid; DIC, di-isopropyl carbodiimide;
DMAP, dimethyl amino pyridine; GPCR, G-protein coupled receptors; HRMS, high
resolution mass spectrometry; LPA, lysophosphatidic acid; NMR, nuclear magnetic
resonance; PPTS, pyridinium-p-toluene sulfonate; TMSBr, trimethyl silyl bromide.
⇑
Corresponding author. Tel.: +1 901 448 6026; fax: +1 901 448 3446.
E-mail address: dmiller@uthsc.edu (D.D. Miller).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.115

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R. Gupte et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7525–7528
OBn
*
O
HO
H2
OBn
O
O
O
b
a
*
P
MeO
P
C
MeO P CH3
MeO
OMe
OMe
2R, 2S
3R,3S
1
O
OH
O
c
O
d
O
C17H33
O
O
P
O
*
P
MeO
*
MeO
4R,4S
5R,5S
O
O
O
C17H33
C17H33
O
O
e
O
f
O
*
*
P
P
HO
NaO
R =R -isomer
S =S -isomer
R-3-CCPA
S-3-CCPA
6R,6S
Scheme 1. Reagents and conditions: (a) (i) THF, n-BuLi (2.5 M in hexane), ꢁ78 °C, 0.5 h; (ii) R-benzyl glycidyl ether (R)/S-Benzyl glycidyl ether (S); (iii) THF, BF3OET2, ꢁ78 °C,
2 h, 68%; (b) PPTS, toluene, reflux, 5 h, 65%; (c) H2, Pd(OH)2/C,MeOH, 82.5%; (d) C17H33COOH, DMAP, DIC, DCM, 18 h, 78%; (e)TMSBr, CH2Cl2, 1 h 53%; (f) 0.005 M NaOH.
For this reason we describe the synthesis and characterization of
both the R-3-CCPA and S-3-CCPA.
The approach used for the synthesis of the two stereoisomers of
3-CCPA is shown in Scheme 1. Dimethylphosphonate derivatives
2R and 2S were generated from compound 1 using n-butyllithium,
BF3ꢀetherate and either the R- or S-isomer of benzylglycidyl ether.
The corresponding 3-carbacyclic analogs 3R and 3S resulted from
treatment with pyridinium p-toluenesulfonate (PPTS). Following
benzyl group deprotection by hydrogenation, the resulting alco-
hols 4R and 4S were converted to oleoyl esters 5R and 5S using
N,N0-diisopropylcarbodiimide (DIC) and dimethylaminopyridine
(DMAP). Final products R-3-CCPA and S-3-CCPA were prepared
by methyl group deprotection and conversion to the corresponding
sodium salts using TMSBr and dilute NaOH, respectively. Optical
rotations were determined by dissolving the compounds in
methanol, to be +7.3° for R-3-CCPA and for S-3-CCPA, the optical
rotation was ꢁ7.9°. Each compound was purified by silica gel
column chromatography and verified by mass spectrometry,
Figure 1. Dose–response relationship of ATX inhibition by LPA, R-3-CCPA,
S-3-CCPA and racemic-3-CCPA analogs.
NMR and HRMS.
Compounds R-3-CCPA and S-3-CCPA were examined for their
ability to block ATX-mediated hydrolysis of FS-3 (Echelon
Biosciences, Inc., Salt Lake City, UT) using a fluorescence resonance
energy transfer-based assay.21 Recombinant ATX (25 nmol) in the
presence of various concentrations of R-3-CCPA, S-3-CCPA, or
LPA 18:1 (positive control) in assay buffer consisting of 1 mM
MgCl2, 1 mM CaCl2, 3 mM KCl, 140 mM aCl, 50 mM Tris–HCl, pH
using WinNonLinÒ 6.1 (Pharsight, Mountain View, CA) was used
to fit experimental data and calculate Ki and Ki0 using the Michae-
lis–Menten equations for competitive, uncompetitive, mixed-
mode, and non-competitive inhibition as we have described in re-
cent work.22–24 Mechanism of inhibition was assigned based on the
lowest averaged percent residuals for each mechanism derived
from curve fitting. Using this procedure R-3-CCPA and S-3-CCPA
were determined to be mixed-mode ATX inhibitors with Ki values
8.0 and 15
tration 1
l
M fatty acid free BSA was added to FS-3 (final concen-
l
M). Assays were carried out in white wall 96-well plates
(Corning Inc., Corning, NY) and the fluorescence (excitation
485 nm, emission 538 nm) was measured at the beginning and
after 2 h of incubation at 37 °C using a FLEX station II plate reader
(Molecular Devices, Sunnyvale, CA). Data were normalized to the
corresponding vehicle control, and the mean standard deviation
of triplicate wells was used to calculate ATX activity as per cent
of vehicle control. The dose–response relationship of ATX inhibi-
tion showed little difference between the R-3-CCPA, S-3-CCPA or
the racemate (Fig. 1). However, R-3-CCPA was approximately
twofold more potent in this assay than S-3-CCPA. The kinetic
mechanism by which R-3-CCPA and S-3-CCPA inhibited recombi-
nant ATX-mediated hydrolysis of FS-3 were determined by varying
the concentration of the substrate (FS-3, ranging from 0.3 to
of 0.8 and 1.6 lM, respectively.
The lack of ligand stereospecificity of the LPA1, LPA2, and LPA3
receptors has been published previously25 but no information of
stereoselective ligand activation for LPA5 is currently available at
the present time. Racemic-3-CCPA has previously been shown to
bean agonist of the LPA5 GPCR.7 Here, we compared the dose–
response curves of LPA5 activation for R-3-CCPA and S-3-CCPA with
that of the racemate. These experiments were performed in B103
cells stably expressing LPA5. Wild type B103 cells do not produce
Ca2+ transients in response to LPA and are widely used as a host cell
for LPA receptor expression studies. B103-LPA5 cells were loaded
with Fura-2AM for 30 min in modified Krebs buffer containing 2%
(v/v) pluronic acid, rinsed with Krebs buffer, and changes in the
intracellular Ca2+ concentration were monitored by determining
20
lM) in the presence of three concentrations of each inhibitor
(0, 0.5 and 2 times the IC50). Simultaneous non-linear regression

R. Gupte et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7525–7528
7527
significantly higher (p <0.05) efficacy than did R-3-CCPA for LPA5-
mediated calcium mobilization at concentrations above 1
lM
(Fig. 2). Thus, the LPA5 receptor shows a slight stereoselectivity for
the S- over the R-stereoisomer which contrasts the weak preference
(ꢂ2-fold) shown by ATX for the R-isomer.
We have previously shown that racemic 3-CCPA inhibited lung
metastasis of B16-F10 melanoma in a mice model. To extend this
observation, the stereoisomers were characterized in this model.4,5
Eight-week-old female C57Bl/6 mice were inoculated with 5 ꢃ 104
melanoma cells via the tail vein and divided randomly into four
groups. The groups then received either saline vehicle, R-3-CCPA,
S-3-CCPA, or racemate (at 0.5 mg/kg intraperitoneally) 30 min
after the B16-F10 inoculation and daily for an additional 10 days.
Animals in all groups were monitored for an additional 10 days
without further treatments. On day 21, all mice were sacrificed
and lungs were dissected, fixed with formalin and the numbers
of black melanoma nodules on the lung surface were counted in
each sample (Fig. 3). All 3-CCPA treated groups (R-3-CCPA, S-3-
CCPA and Racemic-3-CCPA) significantly reduced the number of
lung metastases compared to the vehicle treated group. However,
no statistically significant differences were found between the
3-CCPA treated groups using ANOVA followed by Newman–Keuls
multiple comparison test.
Figure 2. Dose–response relationship of LPA5 mediated.
In conclusion, we have synthesized pure stereoisomers of
3-CCPA and found that they inhibited ATX in vitro and B16-F10
melanoma metastasis in vivo without significant stereochemical
preference. The lack of stereoselectivity is underlined by the equal
the ratio of emitted light intensities at 520 nm in response to
excitation at 340 and 380 nm using a FLEX station II plate reader
(Molecular Devices, Sunnyvale, CA).26 Compound S-3-CCPA showed
Figure 3. Lack of stereoselectivity in lung metastasis of B16-F10 melanoma cells by R-3-CCPA, S-3-CCPA and racemic-3-CCPA analogs in a mouse model. (A) Representative
images show fixed intrathoracic organs including lung lobules with visible nodules on the surfaces in black. Total numbers of nodules were reduced in samples treated with
7R,7S or racemate compared to vehicle. Scale bar is 0.5 cm. (B) Lung nodules of B16-F10 melanoma cells were quantified. The number of lung nodules was significantly
decreased in groups treated with R-3-CCPA, S-3-CCPA, and the Racemic-3-CCPA compared to vehicle. However, no statistically significant differences were found either
*
between the stereoisomers or the racemate. Data represent the mean SEM, n = 6–8 mice. p <0.05 compared to vehicle analyzed by one-way ANOVA followed by Newman–
Keuls multiple comparison test.

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