Pharmacological evaluation of a novel cyclic phosphatidic acid derivative 3-S-cyclic phosphatidic acid (3-S-cPA)

Article abstract of DOI:10.1016/j.bmc.2012.03.060

Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator possessing cyclic phosphate ring, which is necessary for its specific biological activities. To stabilize cyclic phosphate ring of cPA, we synthesized a series of cPA derivatives. We have shown that racemic 3-S-cPA, with a phosphate oxygen atom replaced with a sulfur atom at the sn-3, was a more effective autotaxin (ATX) inhibitor than cPA. In this study, we showed that racemic 3-S-cPA also had potent biological activities such as inhibition of cancer cell migration, suppression of the nociceptive reflex, and attenuation of ischemia-induced delayed neuronal cell death in the hippocampal CA1. Moreover, we synthesized both enantiomers of palmitoleoyl derivative of 3-S-cPA, and found that the chirality of 3-S-cPA is not involved in ATX inhibition. Based on these findings, racemic 3-S-cPA is suggested as an effective therapeutic compound like cPA.

Full text of DOI:10.1016/j.bmc.2012.03.060

Bioorganic & Medicinal Chemistry 20 (2012) 3196–3201
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Pharmacological evaluation of a novel cyclic phosphatidic acid derivative
3-S-cyclic phosphatidic acid (3-S-cPA)
Emi Nozaki a, Mari Gotoh a, Ryo Tanaka b, Masaru Kato b, Takahiro Suzuki b, Atsuo Nakazaki b,
Harumi Hotta c, Susumu Kobayashi b, Kimiko Murakami-Murofushi a,
⇑
a Graduate School of Humanities and Sciences, Department of Life Science, Ochanomizu University, 2-1-1 Ohtsuka, Bunkyo-ku, Tokyo 112-8610, Japan
b Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510, Japan
c Department of Autonomic Neuroscience, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator possessing cyclic phosphate
ring, which is necessary for its specific biological activities. To stabilize cyclic phosphate ring of cPA, we
synthesized a series of cPA derivatives. We have shown that racemic 3-S-cPA, with a phosphate oxygen
atom replaced with a sulfur atom at the sn-3, was a more effective autotaxin (ATX) inhibitor than cPA. In
this study, we showed that racemic 3-S-cPA also had potent biological activities such as inhibition of can-
cer cell migration, suppression of the nociceptive reflex, and attenuation of ischemia-induced delayed
neuronal cell death in the hippocampal CA1. Moreover, we synthesized both enantiomers of palmitoleoyl
derivative of 3-S-cPA, and found that the chirality of 3-S-cPA is not involved in ATX inhibition. Based on
these findings, racemic 3-S-cPA is suggested as an effective therapeutic compound like cPA.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 3 March 2012
Revised 27 March 2012
Accepted 27 March 2012
Available online 3 April 2012
Keywords:
Cyclic phosphatidic acid
3-S-cPA
Autotaxin
Cancer cell migration
Nociceptive reflex
Ischemia-induced delayed neuronal death
1. Introduction
phosphate ester moiety from hydrolysis. These carba-cPAs, as well
as cPA, are potent inhibitors of ATX, cancer cell invasion and
Cyclic phosphatidic acid (cPA) is a naturally occurring phospho-
lipid mediator, which was originally isolated from the myxoamoe-
bae of a true slime mold, Physarum polycephalum, in 1992.1 At
present, cPA has been found in a wide range of organisms, from
slime molds to humans.2,3 cPA has potent biological activities, such
as inhibition of autotaxin (ATX),4,5 invasion and metastasis of
cancer cells,4,5 suppression of nociceptive responses by primary
afferent C-fiber,6 and attenuation of ischemia-induced delayed
neuronal cell death in rat hippocampal CA1 regions.7 Therefore,
cPA is considered to be a promising candidate for developing an
effective therapeutic agent for some diseases, including cancer
and neurodegeneration.
metastasis, and transient ischemia-caused neuronal damage in
the hippocampal CA1 region.7,9,10 In addition, 2ccPA also sup-
presses the nociceptive reflex similarly to cPA.6 Some studies have
shown that replacing a phosphate oxygen atom with a sulfur atom
results in increased stability to nuclease-mediated and base-cata-
lyzed hydrolysis in nucleic acids.11 Therefore, we have prepared
the sulfur analogs of cPA, that is, racemic 3-S-cPA 1a–d with fatty
acid moieties of 16:1, 18:0, 18:1 or 16:0 (Fig. 1), and investigated
their action toward ATX.12 Our results showed that they have a
similar inhibitory effect on ATX as naturally occurring cPA, and
we presume that numerous biological functions are conserved
between 3-S-cPA and cPA.
cPA has a unique structure, composed of a cyclic phosphate ring
at the sn-2 and the sn-3 positions of the glycerol backbone, which
is required for some biological activities.8 2-O-carba-cPA (2ccPA)
and 3-O-carba-cPA (3ccPA) are derivatives of cPA in which one of
the phosphate oxygen atoms, either at the sn-2 or the sn-3 position,
is replaced with a methylene group in order to protect the cyclic
In this study, we examined the effects of racemic 3-S-cPA on the
following biological activities, (1) inhibition of cancer cell migra-
tion, (2) suppression of the nociceptive reflex, and (3) attenuation
of ischemia-induced delayed neuronal cell death. Subsequently, to
determine the biological activities of enantiopure 3-S-cPA, we syn-
thesized both enantiomers of palmitoleoyl derivative of 3-S-cPA 1a
and studied their effects on ATX activity.
Abbreviation: cPA, cyclic phosphatidic acid; LPA, lysophosphatidic acid; ATX,
autotaxin; 2ccPA, 2-O-carba-cyclic phosphatidic acid; 3ccPA, 3-O-carba-cyclic
phosphatidic acid.
2. Chemistry
We have already synthesized racemic 3-S-cPA 1a–d.12 Accord-
ing to the synthetic method of 3-S-cPA which has been previously
⇑
Corresponding author. Tel./fax: +81 (0) 3 5978 5362.
E-mail address: murofushi.kimiko@ocha.ac.jp (K. Murakami-Murofushi).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.03.060

E. Nozaki et al. / Bioorg. Med. Chem. 20 (2012) 3196–3201
3197
Figure 1. Structure of 3-S-cPA 1a–d.
Figure 2. Effect of racemic 3-S-cPA on MDA-MB-231 cell migration: Maximal
migration activity in the presence of LPC is represented by 100%. Each column
indicates the mean S.E. of triplicate wells and is representative of at least three
independent experiments with similar results. ⁄P < 0.05, ⁄⁄P < 0.01, significantly
different from LPC as determined by one-way ANOVA and Dunnett’s test.
reported by us, both enantiomers of palmitoleoyl derivative of
3-S-cPA 1a were synthesized starting from enantiopure glycidol 2
(Scheme 1). First, commercially available (R)-(+)-glycidol 2 was
esterified with palmitoleic acid using WSC and DMAP. Then, epox-
ide 3a was cleaved with HSCN in acetic acid, and the resulting
alcohol 4a was reacted with salicylchlorophosphite,13 followed
by hydrolysis with TEAB buffer to give H-phosphonate 5a. Finally,
the treatment of 5a with trimethylsilyl chloride formed bistri-
methylsilyl phosphite 6a in situ, which underwent a simultaneous
intramolecular Arbuzov reaction to give cyclic thiophosphate 7a.
After the hydrolysis of 7a in acetonitrile, (R)-(+)-3-S-cPA 1a was
obtained. In the same manner, (S)-(ꢀ)-3-S-cPA 1a was synthesized
from (S)-(ꢀ)-glycidol 2.
3.2. Effect of racemic 3-S-cPA on somato-somatic C-reflex
We have already reported that cPA and 2ccPA suppressed the
nociceptive reflex via C-fibers.6,17 In this study, we examined
whether racemic 3-S-cPA 1a suppresses the nociceptive reflex
induced by the activation of C-fibers. Single shock stimulation of
the myelinated A- and unmyelinated C-afferent fibers of the saphe-
nous nerve (at 10 V with 0.5 ms pulse duration) produced two
distinct A- and C-somatic reflex components in the hind limb elec-
tromyogram (EMG), that is, a short latency (about 10 ms) A-reflex
and a long latency (about 40 ms) C-reflex. Intravenous injection of
0.2 mg/kg racemic 3-S-cPA 1a depressed the C-reflex, but not A-re-
flex. After injection of racemic 3-S-cPA 1a, the C-reflex reached
87 5% of the control (Fig. 3). The effective dose of racemic 3-S-
cPA is comparable to that of racemic 2ccPA (0.2 mg/kg, iv;
83 2.5% of the control); however, the effective dose of racemic
2ccPA was almost 10-fold less than that of natural cPA.6 Therefore
racemic 3-S-cPA and 2ccPA are more potent inhibitors of the
C-reflex than the naturally occurring cPA.
3. Results and discussion
3.1. Effect of racemic 3-S-cPA on MDA-MB-231 cell migration
ATX is a cell motility-stimulating factor that synthesizes lyso-
phosphatidic acid (LPA) by hydrolysis lysophosphatidylcholine
(LPC).14–16 We have previously shown previously that cPA and its
derivatives suppress synthesis of LPA via inhibition of ATX activity,
and as a result, also inhibit cancer cell invasion and metasta-
sis.9,10,17,18 As shown in Figure 2, cell migration was inhibited by
racemic 3-S-cPAs with different fatty acids (1a, b, c and d), to the
same extent (>40%). These results are consistent with those previ-
ously published demonstrating that the nature of the acyl chain of
racemic 3-S-cPA dose not significantly influences the inhibition of
the ATX activity.12
3.3. Effect of racemic 3-S-cPA on delayed neuronal cell death
Recently, we have reported the effects of cPA and 2ccPA on ische-
mia-induced delayed neuronal cell death in the rat hippocampal
Scheme 1. Reagents and conditions: (a) Palmitoleic acid, WSC, DMAP, CH2Cl2, rt; (b) HSCN, AcOH, 72% in two steps; (c) salicylchlorophosphite, pyridine, rt, then TEAB buffer,
62%; (d) TMSCl, Et3N, pyridine, rt, then H2O, MeCN, 81%.

3198
E. Nozaki et al. / Bioorg. Med. Chem. 20 (2012) 3196–3201
Figure 3. Effect of racemic 3-S-cPA on somato-somatic C-reflex: The mean size of
the C-reflex 0–15 min before each injection is expressed as 100%. All subsequent
reflexes are expressed as percentages of the control values. Each column represents
the mean S.E. ⁄⁄⁄P < 0.005, significantly different from control as determined by
paired t-test.
Figure 5. Effect of chirality of 3-S-cPA on ATX activity: The catalytic activity of ATX
in FBS was examined in the presence of (R)-(+)-3-S-cPA 1a (closed circles), (S)-(ꢀ)-
3-S-cPA 1a (open circles), and racemic 3-S-cPA 1a (closed triangles). Each data was
calculated as percent inhibition, by comparison with the respective controls
without any compounds, denoted as 100%. Each data point represents the
mean S.E. of the triplicate wells and is representative of at least three independent
experiments.
CA1 region.7 cPA and 2ccPA significantly attenuates neuronal
damage and decreased glial fibrillary acidic protein (GFAP) accumu-
lation. In the present study, we examined whether racemic 3-S-cPA
1a attenuates neuronal damage in the hippocampal CA1 region and
decreased GFAP accumulation. Figure 4 shows representative histo-
logical pictures of the rat hippocampal CA1 region from coronal
sections taken 5 days after transient occlusion for 8 min. Rats were
treated with either 0.2% fatty acid free bovine serum albumin (BSA)
compared to the vehicle-treated rats (Fig. 4Ac, Bc). These results
demonstrate that 3-S-cPA, as well as cPA and 2ccPA, has a potent
neuroprotective effect.
Subcutaneous administration of racemic 3-S-cPA 1a showed
neuroprotection, as well as cPA and 2ccPA. We suggest the low
molecular weight (507.71 Da) and high lipophilicity (LogP = 5.96)
of racemic 3-S-cPA 1a may allow it to penetrate the blood–brain
barrier BBB, and our results demonstrate that 3-S-cPA, in addition
to cPA and 2ccPA, has potential therapeutic advantages.
vehicle (Fig. 4Aa–c) or 180
l
gꢁkgꢀ1ꢁ5 daysꢀ1 of racemic 3-S-cPA 1a
(Fig. 4Ba–c). The majority of cells in the CA1 region exposed to the
vehicle were histologically damaged, that is, they showed pyknosis
and shrinkage of cell bodies (Fig. 4Ab). Comparatively, the racemic
3-S-cPA 1a treatment induced attenuated neuronal damage in the
hippocampal CA1 region following transient occlusion (Fig. 4Bb).
Transient ischemia induced an increase in anti-GFAP antibody
immunoreactivity as reported previously.19 Treatment with race-
mic 3-S-cPA 1a decreased anti-GFAP antibody immunoreactivity
3.4. Effect of chirality of 3-S-cPA on ATX activity
We synthesized both enantiomers of palmitoleoyl derivative of
3-S-cPA 1a and studied their effects on ATX activity.12 ATX activity
Figure 4. Effect of racemic 3-S-cPA on delayed neuronal death: Representative histological pictures of neurons and astrocytes in the hippocampal CA1 region of the rats
treated with vehicle (0.2% BSA, (A) and racemic 3-S-cPA 1a (180
stained with HE (a, b) and anti-GFAP antibody (c). Arrowheads show the medial and lateral borders of the CA1 region.
l
gꢁkgꢀ1ꢁ5 daysꢀ1; (B) samples were acquired by coronal section on the 5th day after 8 min occlusion, and

E. Nozaki et al. / Bioorg. Med. Chem. 20 (2012) 3196–3201
3199
was measured using the FS-3 substrate, which fluoresces upon
cleavage by ATX. The catalytic activity of ATX in fetal bovine serum
(FBS) was significantly inhibited by (R)-(+)-3-S-cPA 1a, (S)-(ꢀ)-3-S-
cPA 1a and racemic 3-S-cPA 1a (Fig. 5). The ꢂ40% inhibitory effect
To a solution of crude epoxide (S)-3a in AcOH (18 mL) was
added HSCN (1 M in 70% AcOH aq, 18 mL) at room temperature,
then the resulting mixture was stirred at that temperature for
20 min. The reaction mixture was quenched with a saturated aque-
ous solution of NaHCO3. The aqueous layer was extracted with
EtOAc. Combined organic layer was washed with brine, dried over
MgSO4, filtrated and concentrated under reduced pressure. The
residue was purified by silica-gel column chromatography (Hex-
ane/EtOAc = 5/1) to afford the thiocyanate (R)-4a (1.92 g, 72%,
two steps). Colorless oil: TLC, Rf = 0.50 (Hexane/EtOAc = 2/1); IR
(neat) 3463, 3017, 2926, 2855, 2158, 1733, 1458, 1415, 1379,
of (R)-(+)-3-S-cPA 1a on ATX occurred at 10 lM. The dose-response
relationship of ATX inhibition showed no significant difference
between (R)-(+)-3-S-cPA 1a and (S)-(ꢀ)-3-S-cPA 1a, indicating
that the chirality of 3-S-cPA 1a did not affect the degree of ATX
inhibition. This result was consistent with the previous report
that no stereoselective differences were observed between the
enantiopure 2ccPA and 3ccPA with respect to inhibition of
ATX.17,18,20
1215, 1167, 1111, 1016 cmꢀ1 1H NMR (400 MHz, CDCl3) d 5.39–
;
5.30 (m, 2H), 4.30–4.16 (m, 3H), 3.17 (dd, J = 13.5, 4.3, 1H), 3.04
(dd, J = 13.5, 7.1, 1H), 2.37 (t, J = 7.6, 2H), 2.05–1.98 (m, 4H),
1.68–1.59 (m, 2H), 1.37–1.25 (m, 16H), 0.88 (t, J = 6.8, 3H); 13C
NMR (100 MHz, CDCl3) d 173.9, 130.01, 129.66, 112.0, 68.6, 65.8,
37.2, 34.0, 31.7, 29.69, 29.64, 29.09, 29.04, 28.95, 27.19, 27.11,
4. Conclusion
In this study, we clarified the effect of 3-S-cPA on the following
biological functions, which are known to be specific biological
activities of cPA: (1) inhibition of cancer cell migration, (2) sup-
pression of the nociceptive reflex, and (3) attenuation of ische-
mia-induced delayed neuronal cell death. We showed that both
enantiomers of 3-S-cPA inhibit ATX as efficiently as cPA and 2ccPA.
Based on these findings, racemic 3-S-cPA is suggested to be an
effective therapeutic compound for disorders such as cancer and
neurodegeneration.
24.8, 22.6, 14.1; ½a D
ꢃ
+12.3 (c 1.00, CHCl3); HRMS (ESI) Calcd for
C20H35NO3NaS [M+Na]+ 392.2235, Found 392.2324.
5.1.2. H-phosphonate (R)-5a
To a solution of thiocyanate (R)-4a (425 mg, 1.15 mmol) in pyr-
idine (11.5 mL) was added salicylchlorophophite (233 mg,
1.15 mmol) at room temperature, then the resulting mixture was
stirred at that temperature for 2.5 h. The reaction mixture was
quenched with triethylammonium bicarbonate buffer and concen-
trated under reduced pressure. The residue was purified by
reversed-phase chromatography (H2O/MeOH = 1/2) to afford the
H-phosphonate (R)-5a (381 mg, 62%). Colorless oil: TLC, Rf = 0.46
(CHCl3/MeOH/H2O = 60/20/3); IR (neat) 2925, 2854, 2615, 2349,
5. Experimental
5.1. Chemistry
All non-aqueous reactions were performed under an atmo-
sphere of dry argon in oven-dried glassware. Triethylamine (TEA)
and pyridine were distilled from calcium hydride. Other reagents
were used without further purification. Flash column chromatog-
raphy was performed with PSQ 100B (Fuji Silysia Co., Ltd, Japan).
Reversed-phase chromatography was performed with Cosmosil
140C18-PREP (Nacalai Tesque, Inc, Japan). Solvents for chromatog-
raphies are listed as volume/volume ratios. Analytical thin layer
chromatography was performed using commercial silica gel plates
(E. Merck, Silica Gel 60 F254). Infrared spectra (FT-IR) were recorded
on a PerkinElmer Spectrum 100 FT-IR spectrometer. Absorbance
frequencies are recorded in reciprocal centimeters (cmꢀ1). High
resolution mass spectra (HRMS) were obtained from Applied Bio-
systems mass spectrometer (API QSTAR pulsar i) for electrospray
ionization (ESI). Melting points were recorded on Yanaco MP-3S.
1H NMR spectra were acquired at 400 MHz on a JEOL JNM-LD400
spectrometer. Solvent for NMR is used chloroform-d. Chemical
shifts are reported in delta (d) units in parts per million (ppm) rel-
ative to the singlet (7.26 ppm) for chloroform-d. Splitting patterns
are designated as s, singlet; d, doublet; t, triplet; q, quartet; sept,
septet; m, multiplet and br, broad. Coupling constants are recorded
in Hertz (Hz). 13C NMR spectra were acquired at 100 MHz on a JEOL
JNM-LD400 spectrometer. Chemical shifts are reported in ppm rel-
ative to the central line of the triplet at 77.0 ppm for chloroform-d.
2155, 1737, 1456, 1391, 1359, 1218, 1167, 1117, 1056 cmꢀ1 1H
;
NMR (400 MHz, CDCl3) d 12.2 (br s, 1H), 6.96 (d, J = 644, 1H),
5.39–5.30 (m, 2H), 4.74–4.66 (m, 1H), 4.32 (dd, J = 11.7, 5.1, 1H),
4.27 (dd, J = 11.7, 5.8, 1H), 3.37 (dd, J = 13.6, 4.5, 1H), 3.22 (dd,
J = 13.6, 6.3, 1H), 3.08 (qd, J = 7.4, 4.4, 6H), 2.33 (t, J = 7.7, 2H),
2.04–1.98 (m, 4H), 1.65–1.57 (m, 2H), 1.34 (t, J = 7.4, 9H) 1.42–
1.21 (m, 16H), 0.88 (t, J = 7.0, 3H); 13C NMR (100 MHz, CDCl3) d
173.0, 129.88, 129.62, 112.2, 69.8, 64.1, 45.6, 36.7, 33.92, 31.65,
29.60, 29.57, 29.04, 28.99, 28.85, 27.09, 27.05, 24.7, 22.5, 14.0,
8.5; ½a D
ꢃ
+13.9 (c 1.00, CHCl3); LRMS (ESI) Calcd for C20H35NO5PS
[M-Et3NH]ꢀ 432.1974, Found 432.2409.
5.1.3. 3-S-cPA (R)-(+)-1a
To a solution of H-phosphonate (R)-5a (62.0 mg, 0.116 mmol)
in pyridine (1.2 mL) and TEA (0.58 mmol) was added TMSCl
(63 mg, 0.58 mmol) at room temperature, then the resulting mix-
ture was stirred at that temperature for 4 h. The reaction mixture
was concentrated under reduced pressure. To the obtained resi-
due was added MeCN/H2O (v/v = 99/1). The reaction mixture
was concentrated under reduced pressure. The residue was puri-
fied by silica-gel column chromatography (CHCl3/MeOH = 30/1,
containing trace amount of TEA) to afford the triethylammonium
salt of 3-S-cPA (R)-(+)-1a (47.7 mg, 81%). Colorless oil: TLC,
Rf = 0.46 (CHCl3/MeOH/H2O = 60/20/3); IR (neat) 3409, 2978,
2925, 2854, 2602, 2496, 1738, 1475, 1444, 1397, 1383, 1364,
5.1.1. Thiocyanate (R)-4a
1171, 1071, 1035 cmꢀ1 1H NMR (400 MHz, CDCl3) d 12.1 (br s,
;
To a solution of glycidol (R)-(+)-2 (534 mg, 7.21 mmol) and pal-
mitoleic acid (1.83 g, 7.21 mmol) in CH2Cl2 (24 mL) was added
WSC (1.38 g, 7.21 mmol) and DMAP (88 mg, 0.72 mmol) at room
temperature, then the resulting mixture was stirred at that tem-
perature for 5 h. The reaction mixture was quenched with aqueous
citric acid solution. The aqueous layer was extracted with CHCl3
and combined organic layer was washed with a saturated aqueous
solution of NaHCO3 and brine, dried over MgSO4, filtrated and con-
centrated under reduced pressure. The crude epoxide (S)-3a was
used in the next reaction without further purification.
1H), 5.39–5.30 (m, 2H), 4.55–4.47 (m, 1H), 4.29 (dd, J = 11.5, 5.9,
1H), 4.23 (dd, J = 11.5, 5.6, 1H), 3.35–3.24 (m, 2H), 3.10 (qd,
J = 7.2, 4.6, 6H), 2.32 (t, J = 7.7, 2H), 2.05–1.95 (m, 4H), 1.65–1.57
(m, 2H), 1.35 (t, J = 7.2, 9H), 1.39–1.25 (m, 16H), 0.88 (t, J = 6.8,
3H); 13C NMR (100 MHz, CDCl3) d 173.4, 130.01, 129.76, 74.5,
64.5, 45.8, 36.5, 34.1, 31.8, 29.74, 29.71, 29.17, 29.12, 29.11,
28.98, 27.23, 27.17, 24.8, 22.7, 14.1, 8.6; ½a D
ꢃ
+3.76 (c 0.14, CHCl3);
LRMS (ESI) Calcd for C19H34O5PS [M-Et3NH]ꢀ 405.1865, Found
405.2510.

3200
E. Nozaki et al. / Bioorg. Med. Chem. 20 (2012) 3196–3201
5.1.4. 3-S-cPA (S)-(ꢀ)-1a
(0.2 mg/kg) or 2ccPA (0.2 mg/kg). A juglar artery was catheterized
to record the arterial blood pressure and heart rate (Rekuchicoda
WT-685 G, Nihon Kohden, Tokyo, Japan). The ventilation was mon-
itored with a gas analyzer (Respirator SN-480-7, Sinano seis-
akuzyo, Tokyo, Japan) and adjusted to maintain an end-tidal CO2
level at ꢂ3.0%. Body temperature was maintained at ꢂ37.5 °C by
using an automatically regulated heating pad and lamp (Animal
blanket controller ATB-1100, Nihon Kohden, Tokyo, Japan). The
spinal cord was completely transected at the upper thoracic level.
With the rat in a supine position, a branch of the right saphe-
nous nerve innervating the thigh skin was cut at the thigh level
and covered with warm paraffin oil. The central cut end of the seg-
ment of the nerve was placed on bipolar platinum iridium wire
electrodes for electrical stimulation. Single square pulse stimuli
of 10 V with 0.5 ms duration were delivered every 3 s by a digital
electrical stimulator (Nihon Kohden, Tokyo, Japan). Electromyo-
gram (EMG) was recorded from the right leg muscles by inserting
the silver electrodes using the AC preamplifier (Bioelectric ampli-
fier MEG-1200, time set constant at 0.01 s; Nihon Kohden, Tokyo,
Japan). The amplified EMG signals were digitized (micro 1401,
Cambridge Electronic Design, Cambridge, UK). The EMG activity
of the reflex responses induced by single shocks, and was averaged
for approximately 50 trials at 3-s intervals. The size of the reflex re-
sponse was measured as the area under the evoked response and
expressed as the percentage of the control size preceding drug
injection. All data were reported as mean S.E.
3-S-cPA (S)-(ꢀ)-1a was prepared from glycidol (S)-(ꢀ)-2 follow-
ing the same procedures for 3-S-cPA (R)-(+)-1a. ½aꢃD ꢀ4.98 (c 1.00,
CHCl3).
5.2. Biological assays
5.2.1. Migration assay
The migration assay was performed as reported previously with
some modifications.17 Migration of the cells across the 8-
m pore-
l
sized membrane was assessed using Chemo Tx System (Neuro
Probe) according to the manufacturer’s protocol. Briefly, human
breast cancer MDA-MB-231 cells were stained with calcein-AM
(Dojindo, Kumamoto, Japan), and the cell suspension was centri-
fuged and washed with phosphate-buffered saline (PBS). After
washing, the cells were re-suspended in Dulbecco’s modified Ea-
gle’s medium (DMEM; Nissui, Tokyo, Japan) containing 0.1% fatty
acid free bovine serum albumin (BSA; Sigma–Aldrich, MO)
(4 ꢄ 105 cells/mL). To the lower well, 200
lM lysophosphatidyl-
choline (LPC) and 10 lM racemic 3-S-cPA (1a–d) in DMEM con-
taining 0.1% BSA were added. In addition, 25 lL of cell
suspension was added to each upper filter well. Plates were incu-
bated at 37 °C for 8 h, and the medium was carefully removed from
the upper filter. Then, the upper filter was carefully washed with
PBS containing 2 mM ethylendiaminetetraacetic acid (EDTA), and
gently wiped with a cotton swab to remove the non-invaded cells.
The filter was then separated from the microplate, and the fluores-
cence of the migrated cells on each lower well was measured
(emission (em)/excitation (ex): 485/530 nm) with a Cyto Fluor ser-
ies 4000 micro-plate reader (Applied Biosystems, Foster City, CA).
Data were calculated as percent inhibition in comparison to the
respective controls without any compounds, and are shown as
the mean S.E. of triplicate wells.
5.2.5. Transient cerebral ischemia
Transient cerebral ischemia was induced according to the
methods previously described7 with some modifications. The ani-
mals were anesthetized by halothane (3.5% during the induction
of anesthesia; 1.5% during the surgery and experiments) and an
antibiotic (viccllin, 50 mg/kg, intranasal) was administered. An os-
motic pump (Mini-Osmotic Pump Model 2001; Alzet, Cupertino,
CA) containing the vehicle (0.2% fatty acid free BSA) or racemic
3-S-cPA 1a solution was placed under the abdominal skin, and
the solution was continuously administer for 5 days to yield a
5.2.2. ATX inhibition assay
The autotaxin (ATX) inhibition assay was performed according
to a previously described method17 with some modifications.
ATX inhibition activities of (R)-(+)-3-S-cPA 1a, (S)-(ꢀ)-3-S-cPA 1a
and racemic 3-S-cPA 1a were measured using the FS-3 substrate
(Echelon Biosciences, Inc., UT), which fluoresces upon cleavage
by ATX. Fetal bovine serum (FBS; Biowest, FL) was used as the
ATX source and was mixed with various concentrations of com-
concentration of 180
l
gꢁkgꢀ1ꢁ5 daysꢀ1 at a speed of 1
lL/h. The
pump was filled with solutions according to the manufacturer’s
procedure, and was implanted subcutaneously at 35–60 min be-
fore the onset of occlusion. Then, the trachea was incubated,
and respiration was maintained using an artificial respirator
(SN-480-7). Rectal temperature and temporal muscle temperature
were monitored and maintained at approximately 37.5 °C by
using a feedback-controlled heating pad and lamp (ATB-1100).
Vertebral arteries on both sides were permanently ligated, and
common carotid arteries on both sides were transiently occluded
for 8 min. Halothane anesthesia was immediately removed at the
end of transient ischemia, and the respirator was disconnected
after 15 min.
pounds in 0.1% BSA and 1 lM FS-3 in the assay buffer (1 mM CaCl2,
1 mM MgCl2, 5 mM KCl, 140 mM NaCl, 50 mM tris (hydroxy-
methyl) aminomethane (Tris)–HCl, pH 8.0). The fluorescence
intensity (em/ex: 485/530 nm) of each well was measured by a
Cyto Flour series 4000 micro plate reader prior to and after 2 h
incubation at 37 °C. Data were normalized to the vehicle control
after subtraction the before-incubation value and expressed as per-
cent inhibition. All data were shown as mean S.E. of triplicate
wells.
Five days after transient ischemia, the rat was anesthetized by
pentobarbital and perfused transcardially with heparinized saline
followed by 10% formalin in 0.1 M PBS at pH 7.4. The rats were left
in situ at 4 °C for 2 h. Then, the brains were removed, coronally cut
into 3 mm thick slices, and the specimens were embedded in par-
5.2.3. Animals
The experiments were performed with adult male Wistar rats
(body weight, 310–370 g). The rats were provided by the animal
breeding farm at the Tokyo Metropolitan Institute of Gerontology.
This study was approved by the Animal Committee of the
Institution.
affin. Cross-sections of the brain were cut at 6 lm thickness. Brain
sections were stained with hematoxylin and eosin (HE), or immu-
nohistochemically stained with anti-glial fibrillary acidic protein
(GFAP) rabbit antibody (Dako Japan Inc., Tokyo, Japan) to identify
the astrocytes. The dorsal hippocampus, approximately 3.3 mm
posterior to the bregma, was selected as the representative area
for CA1 neurons.
5.2.4. Recording of spinal somato-somatic reflex
The recording and induction of spinal somato-somatic reflexes
was completed according to previously described procedures17
with some modifications. The animals were anesthetized by pento-
barbital (somunopentyl, 50 mg/kg, intraperitoneal (ip); Kyoritsu
Seiyaku Corporation, Tokyo, Japan). A juglar vein was catheterized
for intravenous (iv) administration of racemic 3-S-cPA 1a
5.2.6. Statistical analysis
All values are given as the mean S.E. The data were statisti-
cally analyzed by paired t-test, one-way ANOVA, Dunnett’s test,

E. Nozaki et al. / Bioorg. Med. Chem. 20 (2012) 3196–3201
3201
7. Gotoh, M.; Hotta, H.; Murakami-Murofushi, K. Eur. J. Pharmacol. 2010, 649,
206.
8. Kobayashi, S.; Tokunoh, R.; Shibasaki, M.; Shinagawa, R.; Murakami-Murofushi,
K. Tetrahedron Lett. 1993, 34, 4047.
or nonlinear regression. A P-value of <0.05, <0.01 or <0.005 was
considered statistically significant.
9. Baker, D. L.; Fujiwara, Y.; Pigg, K. R.; Tsukahara, R.; Kobayashi, S.; Murofushi, H.;
Uchiyama, A.; Murakami-Murofushi, K.; Koh, E.; Bandle, R. W.; Byun, H.;
Bittman, R.; Fan, D.; Murph, M.; Mills, G. B.; Tigyi, G. J. Biol. Chem. 2006, 281,
22786.
10. Uchiyama, A.; Mukai, M.; Fujiwara, Y.; Kobayashi, S.; Kawai, N.; Murofushi, H.;
Inoue, M.; Enoki, S.; Tanaka, Y.; Niki, T.; Kobayashi, T.; Tigyi, G.; Murakami-
Murofushi, K. Biochim. Biophys. Acta Mol. Cell Biol. Lipids 2007, 1771, 103.
11. Inoue, N.; Minakawa, N.; Matsuda, A. Nucleic Acids Res. 2006, 34, 3476.
12. Tanaka, R.; Kato, M.; Suzuki, T.; Nakazaki, A.; Nozaki, E.; Gotoh, M.; Murakami-
Murofushi, K.; Kobayashi, S. Bioorg. Med. Chem. Lett. 2011, 21, 4180.
13. Young, R. W. J. Am. Chem. Soc. 1952, 74, 1672.
Acknowledgments
This work was supported in part by the Princess Takamatsu
Cancer Research Fund, the Rational Evolutionary Design of
Advanced Biomolecules (REDS3) Project, Central Sitama Area in
the Program for Fostering Regional Innovation (City Area Type),
and
a Grant-in-Aid for Scientific Research (KAKENHI, No.
22790203) from the Ministry for Education, Culture, Sports,
Science and Technology of Japan.
14. Umezu-Goto, M.; Kishi, Y.; Taira, A.; Hama, K.; Dohmae, N.; Takio, K.; Yamori,
T.; Mills, G. B.; Inoue, K.; Aoki, J.; Arai, H. J. Cell Biol. 2002, 158, 227.
15. Tokumura, A.; Majima, E.; Kariya, Y.; Tominaga, K.; Kogure, K.; Yasuda, K.;
Fukuzawa, K. J. Biol. Chem. 2002, 277, 39436.
References and notes
16. Gotoh, M.; Fujiwara, Y.; Yue, J.; Liu, J.; Lee, S.; Fells, J.; Uchiyama, A.; Murakami-
Murofushi, K.; Kennel, S.; Wall, J.; Patil, R.; Gupte, R.; Balazs, L.; Miller, D. D.;
Tigyi, G. J. Biochem. Soc. Trans. 2012, 40, 31.
17. Nozaki, E.; Gotoh, M.; Hotta, H.; Hanazawa, S.; Kobayashi, S.; Murakami-
Murofushi, K. Biochim. Biophys. Acta Mol. Cell Biol. Lipids 2011, 1811, 271.
18. Nozaki, E.; Gotoh, M.; Hanazawa, S.; Mori, H.; Kobayashi, S.; Murakami-
Murofushi, K. Cytologia 2011, 76, 73.
19. Kagitani, F.; Uchida, S.; Hotta, H.; Sato, A. Jpn. J. Physiol. 2000, 50, 585.
20. Gupte, R.; Siddam, A.; Lu, Y.; Li, W.; Fujiwara, Y.; Panupinthu, N.; Pham, T. C.;
Baker, D. L.; Parrill, A. L.; Gotoh, M.; Murakami-Murofushi, K.; Kobayashi, S.;
Mills, G. B.; Tigyi, G.; Miller, D. D. Bioorg. Med. Chem. Lett. 2010, 20,
7525.
1. Murakami-Murofushi, K.; Shioda, M.; Kaji, K.; Yoshida, S.; Murofushi, H. J. Biol.
Chem. 1992, 267, 21512.
2. Takahashi, Y.; Shimada, Y.; Shioda, M.; Yoshida, S.; Murofushi, H.; Murakami-
Murofushi, K. Cell Struct. Funct. 1993, 18, 135.
3. Kobayashi, T.; Tanaka-Ishii, R.; Taguchi, R.; Ikezawa, H.; Murakami-Murofushi,
K. Life Sci. 1999, 65, 2185.
4. Murakami-Murofushi, K.; Uchiyama, A.; Fujiwara, Y.; Kobayashi, T.; Kobayashi,
S.; Mukai, M.; Murofushi, H.; Tigyi, G. Biochim. Biophys. Acta Mol. Cell Biol. Lipids
2002, 1582, 1.
5. Fujiwara, Y. Biochim. Biophys. Acta Mol. Cell Biol. Lipids 2008, 1781, 519.
6. Kakiuchi, Y.; Nagai, J.; Gotoh, M.; Hotta, H.; Murofushi, H.; Ogawa, T.; Ueda, H.;
Murakami-Murofushi, K. Mol. Pain 2011, 7.